우리는 GL261 룩 세포의 준비에 도움이 제안에 대한 lentivirus 시스템뿐만 아니라 Mahil 라오에 대한 plasmids의 관대한 선물에 대한 박사 조슈아 B. 루빈을 인정하고 싶습니다. 우리는 뇌 종양 연구 (SSBTR), 배로우 신경 재단과 관대한 지원 월러스 재단을 지원하는 학생 주셔서 감사합니다. 동물 실험은 세인트 조셉 병원과 의료 센터의 기관 동물 케어 및 사용위원회에 의해 명시된 지침과 규정에 따라 수행되었다.

<ol>
	<li>Candolfi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathological+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.</a> J Neurooncol. 85, 133-148 (2007).</li><li>Stafford, P., Abdelwahab, M. G., do, K. i. m., Preul, Y., Rho, M. C., M, J., Scheck, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ketogenic+diet+reverses+gene+expression+patterns+and+redudes+reactive+oxygen+species+levels+when+used+as+an+adjuvant+therapy+for+glioma.">The ketogenic diet reverses gene expression patterns and redudes reactive oxygen species levels when used as an adjuvant therapy for glioma.</a> Nutr Metab. 7, (2010).</li><li>Caliper Life Sciences, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Living Image Software Version 4.0. VivoVision Systems. , (2010).</li><li>Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Jouanneau, E., Poujol, D., Gulia, S., Le, M. I., Blay, J. Y., Belin, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+are+essential+for+priming+but+inefficient+for+boosting+antitumour+immune+response+in+an+orthotopic+murine+glioma+model.">Dendritic cells are essential for priming but inefficient for boosting antitumour immune response in an orthotopic murine glioma model.</a> Cancer Immunol Immunother. 55, 254-267 (2006).</li></ol>

이 기사에 무료로 액세스 캘리퍼스 생명 과학 후원됩니다.

셀 inoculum은 0.4 mm의 포켓을 만든 후 두뇌의 표면에서 2.6 mm의 깊이에 들어갈 수 있습니다. 바늘의 적절한 배치와 심도를 보장하기 위해 X - 레이는 C - 암 또는 이와 유사한 X - 선 이미지 가속화 장치를 사용 취할 수 있지만, 이것은 선택 사항입니다. 동물이 제대로 진정되지 않으면 수술의 합병증은 동물 세포 주입하는 동안 이동할 수있는 시점에서 발생할 수 있습니다. 이것은 바늘 트랙에서 셀 믹스 또는 출혈의 누출을 일으킬 수 있습니다. 세포의 누수는 종양 세포의 성장을 이소성됩니다. burrhole이 프로토콜 4 설명한 2.3 mm까지 중간 만든 경우에도 소중히 할 수있는 뇌실를 중요하지 않습니다. 바늘과 세포 확산의 적절한 배치는 2 μl 메틸렌 블루 염색와 마우스를 일으키는 및 주입 염료의 위치를​​ 확인하기 위해 뇌 조직을 해부하여 테스트되었습니다. 이 프로토콜에서 우리는 생체내 이미징 시스템 및 t의 IVIS 스펙트럼을 사용한그는이 악기와 함께 사용하도록 설계 이미지 소프트웨어 (V 4.0) 생활. (캘리퍼스 생명 과학). 생체내 이미징 시스템과 이미지 분석 도구의 모든 비교는 유사한 결과를 얻을하는 데 사용할 수 있습니다. 이러한 시스템은 실험 intracranial 종양의 성장에 따라 기존의 자기 공명 이미징 (MRI)을 통해 몇 가지 이점을 제공합니다. 가장 확실한는 2 악기의 상대적인 비용이다 - 동물 MRI 기계는 훨씬 더 비싼 있으며, 일반적으로 숙련된 기술자 MRI의 서비스를 필요로합니다. 이러한 여기에서 설명한 있었는지와 같은 생체내 이미징의 최종 사용자에 의해 할 수 있습니다. MRI의 데이터 quantitation는 많은 시간과 다소 엄밀하지 않은 상태에서 bioluminescence 데이터는 양적 있습니다. 또한, MRI 이미지는 부종과 종양 세포 이외에 염증을 표시하고 치료 효과에서 종양을 구분하기 어려울 수 있습니다. 이러한 이유로 성장하는 종양의 정확한 용적 측정을 획득하는 것은 도전이 될 수 있습니다. Bioluminescence는 ATP가 필요합니다그러므로 오직 살아있는 종양 세포는 종양의 크기 데이터에 기여하고 있습니다. 기계가 사용할 수있는 경우에도 불구하고, MRI를 몇 가지 이점이 있습니다. 세포는 MRI에 의해 시각이 될 수있는 발광 생물 마커로 표시 될 필요가 없습니다. 사망 시에 tumoral 부종을 시각화하는 능력은 몇 가지 실험 프로토콜에 대한 이점 수 있습니다. 두 기술을 사용할 때 두 기술의 강도가 하나를 사용하면 다른의 사용을 배제하지 않으므로 데이터는 종양의 성장뿐만 아니라 같은 동물의 사망 시에 tumoral 부종과 염증의 존재를 얻을 수 있습니다 연구원 수 있습니다.

<table border="1"><tbody><tr><td> 시약이나 공급의 명칭 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> GL261 - luc2 Bioware 울트라 </td><td> 캘리퍼스 생명 과학 </td><td> GL261 - luc2 </td><td></td></tr><tr><td> Dulbecco의 수정 독수리 중간 (DMEM) </td><td> Invitrogen </td><td> 10313039 </td><td></td></tr><tr><td> Geneticin (G418) </td><td> Gibco (Invitrogen) </td><td> 11811-023 </td><td></td></tr><tr><td> 소태아 세럼 (FCS) </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr><tr><td> Phospate는 버​​퍼 식염수 (PBS) </td><td> Invitrogen </td><td> 70011044 </td><td></td></tr><tr><td> C57BL/6-cBrd/cBrd/Cr 마우스 </td><td> NCI - 프레드릭 </td><td></td><td></td></tr><tr><td> AKWA의 눈물 윤활유 안과 연고 </td><td> Akorn Inc의 </td><td> 17478-062-35 </td><td></td></tr><tr><td> Ketaset (케타민을 하이드로 클로라이드) </td><td> Wyeth </td><td> 11570775 </td><td></td></tr><tr><td> Sedazine (xylazine 염산염) </td><td> Wyeth </td><td> 10031894 </td><td></td></tr><tr><td> 작은 동물 Stereotaxic 악기 </td><td> Kopf 인 스트 루먼트 </td><td> 900 </td><td></td></tr><tr><td> SYS - Micro4 컨트롤러 UltraMicroPump </td><td> 세계 정밀 계측기 </td><td> UMP3 - 1 </td><td> 사용할 수없는 경우 수동으로 달이다 수 있습니다 </td></tr><tr><td> 26 게이지와 10μl 주사기는 바늘 beveled </td><td> 세계 정밀 계측기 </td><td> SGE010RNS </td><td></td></tr><tr><td> Adison 집게 </td><td> 세계 정밀 계측기 </td><td> 500,092 </td><td></td></tr><tr><td> Penfield 해부학자 </td><td> Codman </td><td> 65-1015 </td><td></td></tr><tr><td> 16g 1 ½ 정밀 글라이드 니들 </td><td> Beckton, 디킨슨과 회사 (BD) </td><td> 305,198 </td><td></td></tr><tr><td> 외과 블레이드 핸들 </td><td> BD </td><td> 371,030 </td><td></td></tr><tr><td> 15 사이즈 블레이드 </td><td> BD </td><td> 371,315 </td><td></td></tr><tr><td> 4-0 Vicryl 봉합사 </td><td> Ethicon </td><td> VCP496G </td><td></td></tr><tr><td> 뼈 왁스 </td><td> MEDLINE </td><td> DYNJBW25 </td><td></td></tr><tr><td> Povidine - 요오드 면봉 스틱 </td><td> MEDLINE </td><td> MD93901 </td><td></td></tr><tr><td> D - Luciferin의 칼륨 소금 </td><td> 캘리퍼스 생명 과학 </td><td> 122,796 </td><td></td></tr><tr><td> Forane (Isoflurane) </td><td> 벡스터 </td><td> 1001936060 </td><td></td></tr><tr><td> OPMI Pentero 현미경 </td><td> 칼 자이스 혈구, 주식 회사 </td><td></td><td> 모든 수술 현미경은 충분 </td></tr><tr><td> 선택 마취 시스템과 Xenogen IVIS 스펙트럼 </td><td> 캘리퍼스 생명 과학 </td><td></td><td></td></tr></tbody></table>

intracranial implantation with subsequent 3d in vivo bioluminescent imaging of murine gliomas

마우스 glioma 261 (GL261)은 생체내 모델 시스템에으로 인식되고 인간 glioblastoma multiforme의 (GBM)의 기능 중 많은 recapitulates. 셀 라인은 원래 C57BL / 6 마우스 syngeneic 스트레인 하나에 3 - 메틸 - cholantrene의 intracranial 주입에 의해 유도되었다, 따라서 immunologically 유능한 C57BL / 6 생쥐를 사용할 수 있습니다. 우리가 GL261을 사용하는 동안 다음과 같은 프로토콜은 어떤 intracranial 마우스 종양 모델의 주입 모니터링에 사용할 수 있습니다. GL261 세포는 안정적으로 표현 반딧불 루시페라제 (GL261 룩)으로 설계되었습니다. 우리는 또한 CMV 프로 모터에서 표현 luc2 유전자의 안정적인 transfection하여 밝은 GL261 - luc2 세포주를 만들었습니다. 국립 암 연구소, 프레데릭, MD에서 C57BL/6-cBrd/cBrd/Cr 마우스 (C57BL / 6의 흰둥이 변종)는 검은 피부와 모피로 인해 빛을 감쇠를 제거하는 데 사용되었습니다. 흰둥이 C57BL / 6 마우스의 사용을 통해, 생체내 이미징의 생체내 메신저에서 IVIS 스펙트럼을 사용하여시스템을 노화 것은 주입의 날 (캘리퍼스 생명 과학, Hopkinton, MA)에서 가능합니다. GL261 룩과 GL261 - luc2 셀 라인은 부모 GL261 세포로 생체내 행동 동일하게 나타났다. 인간 GBMs에있는 공유 histological 기능이 마우스 모델 중 일부는 다음과 같습니다 : 종양 괴사, pseudopalisades, neovascularization, 침공, hypercellularity과 염증 1. 이전 주입 동물 stereotactic기구와 절개에 배치 300ml의 intraperitoneal 주사 (50 MG / kg), xylazine (5 밀리그램 / kg)과 buprenorphine (0.05 밀리그램 / kg)에 의해 anesthetized되었다이 이상 메스로 만들어진 두개골 중간선. burrhole는 bregma과 중간선의 오른쪽에있는 2.3mm로 0.1mm 사후되었다. 바늘은 2.6mm의 깊이 3mm와 0.4mm 철회의 깊이로 삽입되었다. GL261 룩이나 GL261 - luc2 전지 (10 7 셀 / ML)의 두 μl는 삼분의 과정을 통해 주입했다. burrhole는 폐쇄되었습니다bonewax과 절개는 봉합했습니다와 함께. stereotactic 주입 따라 발광 생물 세포가 이식 당일부터 감지하고 종양이 IVIS 스펙트럼 악기의 3D 이미지 재구성 기능을 사용하여 분석하실 수 있습니다. 동물 150μg luciferin / 이전 이미지로 kg의 체중 20 분의 피하 주사를 받게됩니다. 종양 부담 시간이 지남에 따라 의미 종양의 bioluminescence를 사용하여 계량입니다. 종양 베어링 생쥐는 병적 상태를 평가하기 위해 매일 관찰되었으며 다음과 같은 현상이 하나 이상 존재하는 경우 euthanized되었습니다 혼수, ambulate에 실패, hunched 자세, 신랑에 실패, 거식증은 체중의&gt; 10 %의 손실 발생. 종양 검시에있는 동물의 모든 분명했다.

Watch this Scientific Journal Video about Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas at JoVE.com

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

C57BL에 GL261 셀 / 6 마​​우스의 Intracranial 이식은 인간 glioblastoma multiforme의의 특징 중 많은 것들을 요점을 되풀이하다 악성 gliomas을 생산하고 있습니다. 우리는 우리가 사용할 수 있도록 안정 루시페라제 표현 GL261 세포를 사용<em&gt; 생체내에</em&gt; 영상은 종양 진행을 따르십시오. 수술 및 3D<em&gt; 생체내에</em&gt; 영상을 보여주 있습니다.

이후 3D로 Intracranial 주입 VIVO에서</emMurine Gliomas의> 발광 생물 이미징

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

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Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

20.4K 조회수.  Barrow Neurological Institute of St. Joseph’s Hospital and Medical Center. 中文

비디오: Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Murine 발광 생물 간장 Tumour 모델

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

연구

의학

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

생체 듀얼 기판 발광 생물 이미징에

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

마우스의 전단 응력 유발 죽상 경화증을 실행을위한 경동맥 혈압계의 주입

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Stereotactic Intracranial 주입 및<em&gt; 생체에</emGlioblastoma의 Multiforme의 마우스 모델 시스템에서 종양 Xenografts의&gt; 발광 생물 이미징

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

재 협착의 연구에 대한 경동맥 스텐트 시술의 쥐과 모형

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

소설 고해상도<em&gt; 생체 내</em종양의 성장과 치료학에 두개 내 구조물의 동적 응답을 공부하기&gt; 이미징 기술

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

에 대한 쥐의 내시경<em&gt; 생체</em&gt; 발암 및 소장의 평가의 멀티 모달 이미징 상처 치유 및 염증

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

혈관 주위 약물 전달 패치의 주입과 대퇴 동맥 와이어 부상의 쥐 모델

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light Sheet Microscopy에 의한 심근 성 심근염의 Murine 모델에서의 백혈구 침윤의 정량적 시각화

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

이후 3D로 Intracranial 주입 VIVO에서 발광 생물 이미징