기금 현재의 연구는 의학 스탠포드 유니버 시티 스쿨, 스탠포드, CA, 미국에서 Cardiothoracic 수술의학과에 대한 포크 연구 기금에 의해 지원되었다. 토바 이어스 Deuse는 독일 심장 학회의 연구 보조금에 의해 투자되었다. 소냐 Schrepfer는 독일 Forschungsgemeinschaft (DFG) (SCHR992/2-1)에서 연구 부여를 받았습니다.

대동맥 스텐트 배포 <ol><li> 5백50-6백g 무게 남자 스프 라그 - 돌리 쥐가 할렌 (인디애나 폴리스, 인디애나, 미국)에서 구입할 수 있습니다. 일반적인 조건 하에서 하우스 쥐, 먹이 표준 쥐 차우와 물 광고 libitum. </li><li> 로 쥐를 마취 isoflurane (2 %)과 케타민 (25 MG / kg). 미세한보기에서 infrarenal 대동맥을 노출 미니 개복술 상단 중간을 수행합니다. </li><li> 신장 동맥의 수준에서 아래 분기점으로, 주변 조직에서 복부 대동맥을 절개하다. 주의 : IVC에서 대동맥을 절개하다 필요 없어. </li><li> 대동맥 혈액 흐름을 중지 microclamps를 사용합니다. 말초 클램프 다음 먼저 근위 클램프를 놓으십시오. </li><li> 작은 가로 절개로 대동맥을 열고 헤파린 (200 세대)과 대동맥을 플러시. </li><li> 대동맥 내피는 2 프랑​​스어 포가티 동맥 embolectomy의 카테터 (박스터 헬스케어, 디어 필드, IL, 미국)의 통로로 denuded 있습니다. </li><li> 모든 인간의 스텐트 8mm와 길이 12mm 사이의 크기, 직경 2.5mm - 3mm를 사용합니다. 주의 : 스텐 트의 직경은 사전 및 사후 스텐트 협착을 피하기 위해 10 분의 1 이상으로 혈관 직경을 초과해서는 안됩니다. 주의 : 동일한 연구 내에 스텐트 길이를 변경하지 마십시오. </li><li> 원하는 직경을 달성하기 위해 적절한 풍선 압력을 사용하여 스텐 트를 배포합니다. </li><li> 작은 대동맥 절개는 9-0 Prolene의 봉합 (Ethicon, Norderstedt, 독일)와 닫힙니다. </li><li> 4-0 Vicryl 실행 봉합 (Ethicon, Norderstedt, 독일)와 레이어의 복부 절개를 닫습니다. 피부 봉합은 아직도 Viacryl를 사용해도 7~14일 이내에 제거하여야한다. </li><li> 동물은 식수 7 일간 intraoperatively Carprofen (6mg/KG) 및 Metamizol를 받았다. </li></ol> 광학 결맞음 Tomography (-10 월) 이미징 OCT 이미지는 M2 OCT 이미징 시스템 (LightLab 이미징 주식 회사, 포드, MA, 미국)와 함께 얻을 수 있습니다. ImageWire은 조직에 빛을 전달하고 신호를 수집하기 위해 이미징 프로브입니다. ImageWire는 0.006로 구성되어 있습니다 &quot;(0.15 ㎜) 광섬유 코어 0.019 최대 OD와 칼집 안에&quot;(0.48 mm). <ol><li> 로 쥐를 마취 isoflurane (2 %)과 케타민 (25 MG / kg). 미세한보기에서 infrarenal 대동맥을 폭로하기 위해 중간 다시 실행 개복술을 수행합니다. </li><li> 근위 대동맥과 장골의 동맥을 모두 클램프. </li><li> 대동맥의 말초 끝에 가로 arteriotomy을 수행, 1ml PBS는 28G 카테터를 이용한 플러시와 OCT의 카테터를 삽입하고 대동맥에 그것을 전달합니다. </li><li> 전동 pullback OCT 영상은 초당 1.0 mm의 pullback 속도로 수행됩니다. </li><li> 초당 15 프레임의 영상을 취득, 이미지는 색상 룩업 테이블로 표시되고 디지털로 저장이됩니다. </li><li> arteriotomy를 닫습니다. </li><li> 4-0 Vicryl 실행 봉합 (Ethicon, Norderstedt, 독일)와 복부 및 incisions를 닫습니다. 피부 봉합은 아직도 Viacryl를 사용해도 7~14일 이내에 제거하여야한다. </li><li> 최대 neointima 형성은 스텐트 이식 후 6 주 내에 개발할 것입니다. </li></ol> OCT 이미지 분석 OCT 측정은 쥐를 기반 인터페이스와 LightLab OCT 이미징 독점 소프트웨어를 사용하여 수행됩니다. <ol><li> 이 시스템의 표준 교정 기법입니다 OCT 이미징 와이어의 반사에 시스템을 보정합니다. </li><li> 추적 루멘 각 1.0 mm 간격으로 수동으로 스텐트 교차 단면 영역 (CSA). </li><li> 루멘 CSA에서 빼는 스텐트 CSA와 같은 상패 크로스 단면적을 계산합니다. 스텐트 CSA (%)로 나누어 상패의 CSA 등 %의 플라크 영역을 계산합니다. 평균 퍼센트 플라크 영역을 계산합니다. </li></ol> OCT 기술의 검증 OCT 결과는 조직 병리학 (그림 1)과 잘 상관 관계. 위에서 설명한대로 Histologic 상패의 CSAs이 계산됩니다. 조직학은 스핀들 모양의 세포의 고밀도 단 몇 mononuclear 염증 세포와 내막 증식을 보여줍니다. 육주 후, stents 완전히 neointimal 과립 조직으로 덮여 있으며, 상패의 CSA가 1.3 ± 0.4 mm 대책 2 2.5mm 스텐트 인치 <img src="/files/ftp_upload/1346/1346fig1tmb.jpg" border="0" /> 그림 1 : OCT (A)와 histological (B, 배율 16X) 배포 후 스텐트 6주 이미지. OCT 이미지에서 얻은 상패의 CSA 결과는 조직 병리학과 잘 상관 관계. 하시기 바랍니다 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/1346/1346fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다.

<ol>
	<li>Langeveld, B., Roks, A. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commentary:+rat+restenosis+models:+means+for+thorough+restenosis+research.">Commentary: rat restenosis models: means for thorough restenosis research.</a> Journal of endovascular therapy. 12 (3), 343-345 (2005).</li><li>Roguin, A., Grenadier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stent-based+percutaneous+coronary+interventions+in+small+coronary+arteries.">Stent-based percutaneous coronary interventions in small coronary arteries.</a> Acute Card Care. 8, 70-74 (2006).</li><li>Post, M. J., Waltenberger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+Is+Beautiful:+A+Miniature+Stent+Model.">Small Is Beautiful: A Miniature Stent Model.</a> Arterioscler. Thromb. Vasc. Biol. 27, 701-702 (2007).</li><li>Langeveld, B., Roks, A. J. M., Tio, R. A., Boven, A. J. v. a. n., Want, J. J. L. v. a. n. d. e. r., Henning, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+abdominal+aorta+stenting:+a+new+and+reliable+small+animal+model+for+in-stent+restenosis.">Rat abdominal aorta stenting: a new and reliable small animal model for in-stent restenosis.</a> Journal of vascular research. 41 (5), 377-386 (2004).</li><li>Lowe, H. C., Chesterman, C. N., Khachigian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+aortic+stenting:+toward+a+simple+model+of+in-stent+restenosis.">Rat aortic stenting: toward a simple model of in-stent restenosis.</a> The American journal of cardiology. 88 (6), 720-721 (2001).</li><li>Groenewegen, R., Harst, R. v. a. n. d. e. r., Roks, R., Buikema, R., Zijlstra, R., Gilst, R. v. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+angiotensin+II+and+angiotensin+II+type+1+receptor+blockade+on+neointimal+formation+after+stent+implantation.">Effects of angiotensin II and angiotensin II type 1 receptor blockade on neointimal formation after stent implantation.</a> International Journal of Cardiology. , (2007).</li><li>de Smet, B. J. G. L., Zijlstra, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+look+at+drug+eluting+stents+with+optical+coherence+tomography.">A look at drug eluting stents with optical coherence tomography.</a> European Heart Journal. 28 (8), 918-919 (2007).</li><li>Pinto, T. L., Waksman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+applications+of+optical+coherence+tomography.">Clinical applications of optical coherence tomography.</a> Journal of interventional cardiology. 19 (6), 566-573 (2006).</li><li>Matsumoto, D., Shite, J., Shinke, T., Otake, H., Tanino, Y., Ogasawara, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neointimal+coverage+of+sirolimus-eluting+stents+at+6-month+follow-up:+evaluated+by+optical+coherence+tomography.">Neointimal coverage of sirolimus-eluting stents at 6-month follow-up: evaluated by optical coherence tomography.</a> European Heart Journal. 28 (8), 961-967 (2007).</li><li>Kawase, Y., Suzuki, Y., Ikeno, F., Yoneyama, R., Hoshino, K., Ly, H. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonuniform+rotational+distortion+between+mechanical+IVUS+and+OCT+using+a+phantom+model.+Ultrasound+in+medicine+&amp;amp;+biology.">Comparison of nonuniform rotational distortion between mechanical IVUS and OCT using a phantom model. Ultrasound in medicine &amp;amp; biology.</a> 33 (1), 67-73 (2007).</li></ol>

모든 쥐가 표준 온도, 습도, 조명 조건 하에서 스탠포드 대학 메디컬 센터 (스탠포드, CA)에서 동물 보호 시설에서 지내게되었고, 쥐 차우 물을 광고 libitum을 제공했다. 조사는 건강의 미국 국립 연구소 (NIH 간행물 번호 85-23, 1996 개정)에 의해 출판 실험실 동물의 관리 및 사용에 대한 가이드를 맞췄나. 연구 프로토콜은 실험실 동물 관리에 행정 패널, 스탠포드 대학에 의해 승인되었습니다.

토끼 장골의 동맥과 돼지 관상 동맥 모델은 가장 자주 스텐트 배치 1, 방사선 및 수술 장비가 필요의 조합에 사용되지만, 동물 주택 용량은 제한하고, 구입의 비용은 높다이다. 쥐 스텐트 모델의 제한 쥐가을 위해 특별히 설계된 stents의 사용에 필요한, 더 많은 혈관 부상 2 결과 금속 대 동맥 비율 및 혈전증 3 인위적으로 높은 발생률이다. 쥐 stenting 모델은 단순하고 저렴, 신속, 정확한 잠복기 모델 4. 로우 외하여 쥐의 대동맥의 직접적인 stenting의 초기보고 이후. 인 스텐트 restenosis의 pathophysiology의 평가에 대해이 모델의 5, 가능성 및 적합성 철저하게 5,6을 보여왔다. 쥐 대동맥의 직경은 physiologic 선박 구조의 중단없이 상업적으로 이용 가능한 stents의 확장을 허용하는 충분한 것입니다. 그것은 그들이 토끼 및 돼지에서 할 같은 혈전 형성, 염증, 그리고 SMC 확산으로 pathophysiological 메커니즘은,이 쥐 모델에서 개발하는 것으로 나타났습니다. 따라서 이러한 모델은 restenosis의 실제 과정의 좋은 표현입니다. OCT 고해상도 이미징 기술은 내막 증식을 평가하는 데 유용합니다. 침투 깊이는 1.5-2 mm이지만, 그 해상도가 intravascular 초음파 (IVUS) 7,8의보다 큰 크기의 순서입니다. IVUS와 OCT를 비교 여러 연구 OCT 현재 스텐트 이식 후 8-10 neointimal 증식을 평가하는 기본 기술입니다 결론을 내린다. 특히 소형 선박 직경과 작은 동물로, 10의 높은 해상도가 그것을 restenosis의 평가에 가장 적합한 이미징 양상을 렌더링. 요약이 비디오는 (1) 쥐 대동맥 stenting이 쉽게 가능합니다 (2) 쥐의 복부 대동맥의 stenting 상업 생산 stents를 테스트하기에 적합하며 (3) OCT 이미지가 - 스텐트의 길이 모니터링을위한 정확하고 우아한 기법을 알게됩니다 restenosis.

imaging in-stent restenosis: an inexpensive, reliable, and rapid preclinical model

restenosis의 잠복기 모델 인 스텐트 restenosis로 연결하고 기존 및 미래의 약물 eluting stents의를 최적화하는 pathophysiological 프로세스를 해명하기 위해 필수적입니다. 항체 및 유전자 변형 및 녹아웃 종자의 다양한 쥐가에서 사용할 수 있습니다. 따라서, 쥐에서 인 - 스텐트 restenosis을위한 모델은 pathobiological 및 pathophysiological 연구 편리한 것입니다. 이 비디오에서는, 우리는 전체 절차를 제시하고 높은 처리량을 스텐트 연구에 적합한 쥐를 스텐트 모델 피트가 - 폭포. 우리는 스텐트 전개의 수술 절차와 OCT의 가장 우아한 기술 (광학 결맞음의 Tomography)를 사용 - 스텐트 restenosis의 평가를 표시합니다. 이 기술은 플라크의 CSAs 평가에서 높은 정확도를 제공합니다 (크로스 섹션 영역)와 특별하고 시간이 소요될 삽입과 sectioning 기술을 필요로 histological 섹션과 잘 연결합니다. OCT 영상 추가 조직학을 사용하여 한 시간 스냅샷에 비해 동일한 동물 사이에서 - 스텐트 restenosis의 발전 세로 모니터링을 허용합니다.

Watch this Scientific Journal Video about Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model at JoVE.com

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

이 비디오에서 - 스텐트 restenosis 개발 pathobiological과 pathophysiological 프로세스를 연구하기 위해 잠복기 저렴하고 안정적​​인 모델을 사용하는 방법을 보여줍니다. OCT (광학 결맞음 Tomography)와 OCT 이미지 분석을 사용하여 생체내 모니터링 세로도 증명하고 있습니다.

이미징에서 스텐트 - Restenosis : 저렴한, 신뢰성 및 빠른 잠복기 모델

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and ...

imaging-stent-restenosis-an-inexpensive-reliable-rapid-preclinical

Nanyang Technological University

Research

JoVE Journal

Biology

13.7K 조회수.  Stanford University School of Medicine. 이 비디오에서 - 스텐트 restenosis 개발 pathobiological과 pathophysiological 프로세스를 연구하기 위해 잠복기 저렴하고 안정적​​인 모델을 사용하는 방법을 보여줍니다. OCT (광학 결맞음 Tomography)와 OCT 이미지 분석을 사용하여 생체내 모니터링 세로도 증명하고 있습니다.

비디오: Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

Microfluidic 장치의 저렴한 제작에 대한 PDMS의 비 플라즈마 본딩

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

연구

생물학

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

조립, 로딩 및 분석 초원 심 분리기 샘플 셀 정렬

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

잠복기 X-선 계산 된 단층 데이터의 3D 인쇄 설정

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

비행 성능의 정확하고 빠른 측정을위한 개선 된 방법에<em&gt; 초파리</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

신속한 분석 및 형광 현미경 이미지의 탐사

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

마이 오신 중쇄 면역를 사용하여 쥐의 근육 크로스 섹션에서 근육 섬유 인구 분석을위한 신속한 자동화 프로토콜

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac resynchronization therapy (CRT), and LBBB has become one of the important predictors for CRT response. The conventional tachypacing-induced HF model has several major limitations, including absence of stable LBBB and rapid reversal of left ventricular (LV) dysfunction after cessation of pacing. Hence, it is essential to establish an optimal model of chronic HF with isolated LBBB for studying CRT benefits. In the present study, a canine model of asynchronous HF induced by left bundle branch (LBB) ablation and 4 weeks of rapid right ventricular (RV) pacing is established. The RV and right atrial (RA) pacing electrodes via the jugular vein approach, together with an epicardial LV pacing electrode, were implanted for CRT performance. Presented here are the detailed protocols of radiofrequency (RF) catheter ablation, pacing leads implantation, and rapid pacing strategy. Intracardiac and surface electrograms during operation were also provided for a better understanding of LBB ablation. Two-dimensional speckle tracking imaging and aortic velocity time integral (aVTI) were acquired to validate the chronic stable HF model with LV asynchrony and CRT benefits. By coordinating ventricular activation and contraction, CRT uniformed the LV mechanical work and restored LV pump function, which was followed by reversal of LV dilation. Moreover, the histopathological study revealed a significant restoration of cardiomyocyte diameter and collagen volume fraction (CVF) after CRT performance, indicating a histologic and cellular reverse remodeling elicited by CRT. In this report, we described a feasible and valid method to develop a chronic asynchronous HF model, which was suitable for studying structural and biologic reverse remodeling following CRT.

The establishment of a chronic asynchronous heart failure (HF) model by rapid pacing combined with left bundle branch ablation is presented. Two-dimensional speckle tracking imaging and aortic velocity time integral are applied to validate this stable HF model with left ventricular asynchrony and the benefits of cardiac resynchronization therapy.

심장 재 동기화 치료 왼쪽된 번들 지점 제거 및 빠른 속도 의해 유도 된 비동기 심장 마비 모델에서의 장점

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

애벌레 Zebrafish를 사용 하 여 창 자 환승 시간의 높은 처리량 측정

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.