Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.

참고 : 모든 동물의 절차는 텍사스 건강 과학 센터 샌 안토니오 대학의 기관 동물 케어 및 사용위원회에 의해 승인 도착 도착 가이드 라인과 일치했다되었다. 1. 두피 준비를위한 마취 <ol><li> 마취를 유도하는 산소와 혼합 2~3% 이소 플루오 란으로 유도 챔버에 놓고 마우스. 마우스가 유도로 호흡 속도 감소를 관찰합니다. 마우스가 코 콘으로 이동 준비가되어 있는지 여부를 확인하려면 마우스의 발을 꼬집어. 주 : 마취 레벨이 어떤 생체 제조에서 중요한 단계 및 관리에 글로벌 허혈 발생할 수준을 유도하지 않도록주의해야한다. </li><li> 마우스가 충분히 마취되면, 수술 / 이미징 플랫폼에 동물을 전송하고 노즈콘에서 마우스의 코를 배치하고 마취 상태를 유지 1-1.2 % 이소 플루오 란을 적용한다. 마우스가 온도 공동에 누워 있는지 확인ntrolled 가열 패드 나머지 절차 내내 체온 (37 ° C +/- 0.5 ° C)를 유지한다. 마취 동안 건조를 방지하기 위해 눈을 통해 수의사 연고를 놓습니다. </li><li> 시스템과 함께 제공되는 꼬리 클립이나 발을 사용하여 산소 포화도 시스템을 사용하여 마우스의 생리 모니터. 호흡 속도가 50-65 호흡 / 분 사이에 유지되어 있는지 확인합니다. 동물의 장기 생존을 보장하기 위해 97~98% 사이에 유지되는 BPM과 산소 포화도 300-450 사이에 심장 박동이 남아 있는지 확인합니다. </li><li> 마우스가 적절하게 마취되면, 전기 면도기를 사용하여 두개골을 통해 머리를 면도 에탄올 면봉 다음 잔류 머리와 betadine 깨끗를 제거합니다. 무균 수술 환경을 보장하기 위해 세 번에이 절차를 반복합니다. </li></ol> 2. 수술 <ol><li> 완전히 청소하고 면도 두피로, 두​​개골 fissu을 나타 내기 위해 마우스의 두피에 5mm의 절개를입술과 브레 그마를 찾습니다. </li><li> 두개골 상부에 남아있는 근막을 제거하기 위해 멸균면 주걱을 사용합니다. </li><li> 브레 그마의 정위 좌표를 사용하여 두정 피질 위에 놓인 뼈 조직 접착제로 맞춤 제작 한 스테인레스 스틸 링 (그림 1) 접착제 : 측면 -1 -3 mm이고 : 2~4mm을. 주 : 접착제는 일반적으로 뼈가 상 링의 배치 후 2 분 이내에 설정한다. </li><li> 마우스를 안정화 및 이미징 동안 움직임을 방지하도록 정위 홀더 (도 2)에 링 부착합니다. </li><li> 수술 학년 해부 현미경, 천천히 속도 제어 DREMEL 같은 도구 (마이 징거 3.9 mm의 드릴 비트)가 드릴 된 바와 같이 지역 수준을 유지해야하기을 사용하여 두개골에 1-2mm 섹션을 드릴. 지그재그 패턴을 이용하여이를 달성. 열을 방지하기 위해 구축, 낮은에 드릴 속도를 설정하고 자주 휴식을 취. </li><li> 두개골 두개골 모양 시니가 될 때, 숱이 계속얇아진 표면 수준을 유지하기 위해 같은 지그재그 패턴을 이용하여 메스 블레이드를 이용하여 두개골의 뇌 두개골의 얇은 층의 제거를 용이하게 매끄럽게한다. 한 번에 뼈의 얇은 층을 제거하는 가벼운 압력으로 작은 선형 스트로크을 메스 블레이드의 팁을 사용. 혈관이 명확하게 해부 현미경을 통해 볼 때까지이 작업을 계속합니다. </li><li> 실험을 통해 천공 또는 씨닝 공정 동안에 두개골을 어기면, 인해 기저 피질 가능성 손상 동물을 안락사. 주 : 마우스의 두개골은 대략 300 μm의 두께를 얇게되어 두 치밀골 층 (외부 하나는 내부 층)과 치밀골의 두 층 사이에 개재 해면골의 층으로 구성된다. 외부 치밀골의 층과 해면골의 대부분이 남아 치밀골의 대략 50 μm의 층에 생성 5mm 시추 지역은 제거된다 (도 2b 참조). Visualiz혈관의 ATION 최종 그대로 얇게 두개골이 약 50 μm의 두께를 있는지 확인합니다. 이 두께로 얇아 때 두개골은 여전히​​ 그러므로 존재한다. </li></ol> 3. 현미경 셋업 <ol><li> 목표 인버터가 거꾸로 현미경 시스템 (종래의 공 촛점 또는 두 광자 시스템)를 사용합니다. 주 : 표준 직립 현미경을 이용하는 것도 가능하다. 제한 요소는 무대와 목표 사이의 공간이 될 것입니다. 무대에 대한 수정이 설정을 수행 할 필요가있을 수있다. </li><li> 옆 현미경의 기초를 위치 맞춤 제작 단계에 수술 / 영상 플랫폼을 고정합니다. 주 : 플랫폼 대물 하에서 수술 / 이미징 플랫폼의 수직 이동을 허용하도록 실험실 잭을 이용하여 이루어진다. 실험실 잭은 네 개의 원통형 기둥에 고정 플레이트에 장착된다. (그림 2 참조). </li><li> 20X를 포함하는 목표 인버터를 배치두개골 창을 통해 목표. 현미경의 접안 렌즈를 통해 보면 두개 창을 찾아 이미징 영역에서 목표 위치를 외부 광원을 사용한다. 주 : 촬상 영역이 혈관의 존재에 의해 표시 될 것이다. </li><li> 수성 목표를 들어, 인공 뇌척수액 (ACSF)를 사용하여 매체로 인해 잠재적으로 (30 mM의 KCl을 130 mM의 염화나트륨을 200 mM의 NaHCO3를; 30 mM의 HEPES, 100 mM의 포도당 KH 2 PO 4 12 mM의) 영상 중 두개골 공동으로 누설 (그림 3). </li></ol> 4. 로즈 벵갈 염색 준비, 관리 및 뇌졸중의 유도 <ol><li> 인공 뇌척수액 (ACSF) 로즈 벵골의 신선한 20 ㎎ / ㎖ 솔루션을 준비; 필터링 및 관리하기 전에 소독. 재사용 또는 혼합 된 후에 로즈 벵갈 보관하지 마십시오. 각 실험에 대한 새로운 솔루션을합니다. </li><li> 로즈 벵갈의 동안의 0.1 ml의 꼬리 정맥 주입을 줘561 nm의 레이저를 두개 창 통조림 것은 용액의 주입을 충분히 보장한다. 참고 : 장미 벵골은 뇌의 혈관에 주입 한 후 5 초 이내에 가시화 될 것이다. 전체 용기는 로즈 벵갈로 가득해야합니다. </li><li> 벵갈 로즈 염료의 적절한 주입에 이어 병변 특정 부피의 재현성을 보장하기 위해 용기 직경 (40-80 μm의)에 기초하여, 혈전증 적절한 용기를 선택한다. 혈액 흐름의 방향을보고 동맥과 정맥 사이에 차별화 : 동맥 작은 직경의 혈관에 더 큰 직경에서 이동, 혈관 직경이 큰 혈관에 작은에서 이동합니다. 로즈 벵갈가 주입되면이 쉽게 시각화하여 수행됩니다. </li><li> 다음과 같이 현미경 설정을 변경 : <ol><li> 지속 시간을 늘립니다. 참고 :이 사용되는 현미경 시스템에 따라 달라질 수 있습니다. </li><li> 100 %로 레이저 파워를 높입니다. </li><li> 를 사용하여 1 프레임 / sec로 시계열 화상 모아서최대 스캔 속도. </li></ol></li><li> 안정된 혈전이 혈관 내에서 형성 될 때까지 마우스를 스캔합니다. 참고 :이 일반적으로 연속 스캔의 5 분 이내에 달성된다 (그림 4 참조). </li><li> 벵갈 로즈를 이용하여 혈전 형성의 유도에 따라, 다시 해부 현미경으로 촬상 영역에서 마우스를 제거한다. 조심스럽게 두개골 두개골에서 스테인리스 스틸 링을 제거합니다. 출혈의 두개골 창을 검사합니다. 출혈이 발생하면, 실험 종료. </li><li> 두개골을 통해 절개를 닫 6.0 모노 필라멘트 봉합사를 사용합니다. 감염을 방지하기 위해 봉합 라인을 따라 항생제 연고를 놓습니다. Buprenex (0.05 ㎎ / ㎏) 통증 관리를위한 3 일 동안 피하 매 12 시간을 주입한다. </li><li> 완전히 깨어 자유롭게 이동 될 때까지 마취에서 제거 다음 복구 챔버에 마우스를 돌려줍니다. </li><li> 나중에 추가 조사를 위해 깨끗한 케이지에 마우스를 돌려줍니다. </li></ol> (5). 이후의 일에 종 이미징 <ol><li> 이후 일 이후 photothrombosis에 종 이미징을 수행하기 위해 다음과 같은 방법을 사용합니다. <ol><li> 방법 1 섹션에 기재된 바와 같이, 마우스를 마취. </li><li> 적절한 마취되면 미리 드릴 된 촬상 필드를 덮는 피부를 다시 봉합하는 임의의 잔류를 제거하여 두피를 다시 연다. </li><li> 두개골 상부에 남아있는 근막을 제거하기 위해 멸균면 주걱을 사용합니다. </li><li> 이전 이미지 필드를 덮는 뼈 조직 접착제 주문품 스테인리스 링 (도 1)을 붙인다. </li><li> 마우스를 안정화 및 이미징 동안 움직임을 방지하도록 정위 홀더 (도 2)에 링 부착합니다. </li><li> 이전에 얇게 두개골의 기초가되는 혈관을 찾습니다. 이전에 유도 된 혈전의 존재를 확인하기 위해 FITC 덱스 트란의 꼬리 정맥 주사를 사용한다. </li><li> 의를 닫 6.0 모노 필라멘트 봉합사를 사용하여두개골을 통해 정밀도 범위. 감염을 방지하기 위해 봉합 라인을 따라 항생제 연고를 놓습니다. Buprenex (0.05mg / kg) 통증 관리를위한 3 일 동안 피하 매 12 시간을 주입한다. </li><li> 완전히 깨어 자유롭게 이동 될 때까지 마취에서 제거 다음 복구 챔버에 마우스를 돌려줍니다. </li></ol></li></ol> 스트로크 유도 6. 확인 (사후) <ol><li> 연구의 결론에서,도 5에 도시 된 바와 같이, 2,3,5- 트리 페닐 테트라 졸륨 클로라이드 (TTC) 염색을 이용하여 심 박출량을 확인. 전체 방법은 12에서 찾을 수있다. </li></ol>

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Y., Rafie, C., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topological+basis+for+the+robust+distribution+of+blood+to+rodent+neocortex.">Topological basis for the robust distribution of blood to rodent neocortex.</a> Proceedings of the National Academy of Sciences. 107, 12670-12675 (2010).</li><li>Nishimura, N., Rosidi, N. L., Iadecola, C., Schaffer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+collateral+flow+after+occlusion+of+a+single+cortical+penetrating+arteriole.">Limitations of collateral flow after occlusion of a single cortical penetrating arteriole.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 30, 1914-1927 (2010).</li><li>Nishimura, N., Schaffer, C. B., Friedman, B., Lyden, P. D., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetrating+arterioles+are+a+bottleneck+in+the+perfusion+of+neocortex.">Penetrating arterioles are a bottleneck in the perfusion of neocortex.</a> Proceedings of the National Academy of Sciences. 104, 365 (2007).</li><li>Blum, S., 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=Memory+after+silent+stroke:+Hippocampus+and+infarcts+both+matter.">Memory after silent stroke: Hippocampus and infarcts both matter.</a> Neurology. 78, 38-46 (2012).</li><li>Heinsius, T., Bogousslavsky, J., Van Melle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+infarcts+in+the+middle+cerebral+artery+territory+Etiology+and+outcome+patterns.">Large infarcts in the middle cerebral artery territory Etiology and outcome patterns.</a> Neurology. 50, 341-350 (1998).</li><li>Wardlaw, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+causes+lacunar+stroke.">What causes lacunar stroke.</a> Journal of Neurology, Neurosurgery &amp; Psychiatry. 76, 617-619 (2005).</li><li>Inoue, Y., 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=Ischemic+stroke+under+anticoagulant+therapy].+Rinsho+shinkeigaku.">Ischemic stroke under anticoagulant therapy]. Rinsho shinkeigaku.</a> Clinical Neurology. 50, 455-460 (2010).</li><li>Tiannan Wang, W. C., Xie, Y., Zhang, W., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+Volume+of+the+Focal+Cerebral+Ischemic+Lesion+through+Photothrombosis.">Controlling the Volume of the Focal Cerebral Ischemic Lesion through Photothrombosis.</a> American Journal of Biomedical Sciences. 2, 33-42 (2009).</li><li>Head, B. P., Patel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+and+brain+protection.">Anesthetics and brain protection.</a> Current Opinion in Anaesthesiology. 20, 395-399 (2007).</li><li>Kirsch, J. R., Traystman, R. J., Hurn, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+and+cerebroprotection:+experimental+aspects.">Anesthetics and cerebroprotection: experimental aspects.</a> International Anesthesiology Clinics. 34, 73-93 (1996).</li><li>Koerner, I. P., Brambrink, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+protection+by+anesthetic+agents.">Brain protection by anesthetic agents.</a> Current Opinion in Anaesthesiology. 19, 481-486 (2006).</li><li>Gelb, A. W., Bayona, N. A., Wilson, J. X., Cechetto, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propofol+anesthesia+compared+to+awake+reduces+infarct+size+in+rats.">Propofol anesthesia compared to awake reduces infarct size in rats.</a> Anesthesiology. 96, 1183-1190 (2002).</li><li>Bhardwaj, A., Castro, I. A., Alkayed, N. J., Hurn, P. D., Kirsch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetic+choice+of+halothane+versus+propofol:+impact+on+experimental+perioperative+stroke.">Anesthetic choice of halothane versus propofol: impact on experimental perioperative stroke.</a> Stroke; A Journal Of Cerebral Circulation. 32, 1920-1925 (2001).</li><li>Barretto, R. P., Messerschmidt, B., Schnitzer, M. J. <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+fluorescence+imaging+with+high-resolution+microlenses.">In vivo fluorescence imaging with high-resolution microlenses.</a> Nature Methods. 6, 511-512 (2009).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

인간의 응용 프로그램에 동물 실험에서 스트로크 병태 생리를 번역 할 수있는 능력은 실패로 고생하고있다. 그러나, 이러한 모델 photothrombosis 같은 동물 모델의 사용은 개선 된 뇌졸중 병태 생리에 대한 이해와 스트로크 다음 신경 보호를 제공하기위한 새로운 치료 적 접근법의 탐색을 허용한다. 작은 대뇌 피질의 스트로크와 photothrombotic 모델에 의해 생성 microinfarctions은 높은 유병률을 가지고 있?...

바로 로즈 벵갈 유도 다음과 같은 생체 내 세포 반응의 기술 설명을 허용 시각화는 그대로 마우스에 photothrombosis. 로즈 벵골 (4,5,6,7- 테트라 클로로 -2 &#39;, 4&#39;, 5 &#39;, 7&#39;- tetraiodofluorescein)는 동물 모델에서 허혈성 뇌졸중 (마우스 및 래트)을 유도하기 위해 사용되는 감광성 염료이다. 564 nm의 레이저 광에 얇게 두개골을 통해 꼬리 정맥을 통해 조사 및 후속 RB의 볼 루스 주입 후, 혈전 생리적 뇌졸중을 일으키는 한 유도된다. 이 방법은 원래 1977 년에 로젠과 엘 Sabban 설명하고, 이후 1980 년대 중반 1, 2에서 왓슨에 의해 채택되었다. 요컨대, 로즈 벵갈이어서 조직 인자, 혈액 응고 캐스케이드의 개시제를 활성화 반응성 산소 종의 생성을 생성 녹색 여기 광 (우리의 경우에 561 nm의 레이저)을 조사한다. 응고 캐스케이드의 유도는 레 허혈성를 생산임상 스트로크 3 병리학 관련 이온. 스트로크는 뉴런, 아교 세포, 내피 세포 및 면역계 세포 등 다양한 종류의 상호 작용으로 인해 복잡한 기전을 갖는다. 최고의 기술을 선택하는 것은 특정 세포 프로세스가 여러 고려 사항을 필요로 공부한다. 실험 방법은 세 개의 범주에 폭넓게 나뉘어 시험 관내에서, 각각 갖는 장단점 생체 내 및 실리코 체외 연구는 자연 환경으로부터 세포를 제거하는 주요 단점이 있으므로 그대로 볼 효과를 재현 할 수있다. 살아있는 동물. 생체 기법 증가 병진 의미와 질환 상태의 개선 실험 복제를 제공한다. 실리코 일반적 질환 또는 세포 처리의 컴퓨터 모델링을 의미하며, 점점 시험을위한 잠재적 약물 상호 작용을 연구하는데 이용하면서PLE, 수집 된 정보는 여전히 세포 나 조직 생활에서 테스트해야합니다. 실험실 설정에서 뇌졸중의 이상적인 모델은 인구에서 본 것과 유사한 병리학 적 특징을 설명한다. 인구 뇌졸중 일반적인 생리적 특성이 있지만, 손상 발생의 종류에 따라 많은 차이도있다. 인구의 스트로크는 다양한 경색 볼륨뿐만 아니라 각 병리에 관한 메커니즘의 차이에 그 결과 크고 작은 혈관 폐색, 출혈 병변과 동맥이나 심장 - 색전증 동맥에 발생합니다. 뇌졸중 동물 모델을 이용하는 이점은 인간 스트로크 특성을 모방 재현성 경색의 발생이다. 중간 대뇌 동맥 폐색 (색전이나 혈관 필라멘트 방법)하는 모델 말단 MCAO 및 photothrombosis 모델 : 가장 일반적인 동물 스트로크 모델은 사용 동맥 폐쇄를 포함한다. 장점각 모델의 D 단점은 (4 및 5 참조) 다른 곳에서 검토되고있다. 글로벌 허혈성 모델 (MCAO), 수행 상대적으로 쉬운 초점 뇌졸중 모델보다 인간의 스트로크에 덜 관련이있다. 또한, 이러한 방법은 재현성 뇌 경색 병변을 유도 매우 다양하다. 실험이 MCAO 모델을 통해 분명한 이점을 제공 아니라 자신의 실험을 제어로 photothrombosis 모델만큼 높은 재현성이다. 그러나, 미세 혈관 비방에 모델은 최소한의 허혈성 주변부, 세포 -6,7- 살릴 것으로 생각되는 영역을 표시하기 위해 설명되었다. 또한 vasogenic 부종 및 세포 독성 부종 형성은 또한 이미징 영역의 다음 조사를 유도 할 수있다. 이러한 제한에도 불구하고 기술은 스트로크 8, 9, 10, 11 다음의 많은 생리적 과정에 새로운 통찰력을 제공했다.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma</td>
			<td>330000</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Anesthetic</td>
			<td>MWI Veterinary Supply</td>
			<td>088-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond</td>
			<td>1469SB</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>aCSF&#160;</td>
			<td>126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 26 mM NaHCO3 (pH 7.4).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td>Bioindustrial Products</td>
			<td>500-410</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors 14 cm</td>
			<td>Bioindustrial Products</td>
			<td>12-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont High Tech #5 style, straight</td>
			<td>Bioindustrial Products</td>
			<td>TWZ-301.22</td>
			<td></td>
		</tr>
		<tr>
			<td>LabJack 132X80</td>
			<td>Optosigma Co</td>
			<td>123-6670</td>
			<td></td>
		</tr>
		<tr>
			<td>Platform for Labjack 8X 8</td>
			<td>Optosigma Co</td>
			<td>145-1110</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear bar holder from stereotaxic setup</td>
			<td>Stoelting/Cyborg</td>
			<td>51654</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispomed Labvent Rodent anesthesia machine</td>
			<td>DRE, Inc.</td>
			<td>15001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tech IV Isoflurane vaporizer</td>
			<td>DRE, Inc.</td>
			<td>34001</td>
			<td></td>
		</tr>
		<tr>
			<td>F Air Canister</td>
			<td>DRE, Inc</td>
			<td>80120</td>
			<td></td>
		</tr>
		<tr>
			<td>Bain circuit breathing tube</td>
			<td>DRE, Inc</td>
			<td>86111B</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent adapter for bain tube</td>
			<td>DRE, Inc</td>
			<td>891000</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 regulator for oxygen tanks</td>
			<td>DRE, Inc</td>
			<td>CE001E</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent induction chamber</td>
			<td>DRE, Inc</td>
			<td>15004C</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethicon Silk 6-0; 18 in with P-3 needle</td>
			<td>Suture Express</td>
			<td>1639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective inverter Optical Adapter</td>
			<td>LSM technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foredom drill Dual voltage 110/120</td>
			<td>Foredom</td>
			<td>134.53</td>
			<td></td>
		</tr>
	</tbody>
</table>

rose bengal photothrombosis by confocal optical imaging in vivo: a model of single vessel stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

이 방법의 목적은 561 nm의 레이저 광과 두개골의 박형화 꼬리 정맥을 통해 조사 및 후속의 RB 루스 주사 다음 동물 모델에서 허혈성 뇌졸중 (마우스 및 래트)을 유도했다. 도 4의 이미지는 0, 1, 1.5, 2 분으로 조사 영역의 다음 단일 용기 내 혈전 형성의 진행을 보여준다. 혈전 형성하기 전에 전체 선박 로즈 벵갈 흐르는 확보로 인해 흰색입니다. 선박의 조사의 유도에 따라이 선박의 부분?...

Watch this Scientific Journal Video about Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke at JoVE.com

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

공 초점 광학 이미징에 의해 로즈 벵갈 Photothrombosis<em&gt; 생체</em&gt; : 단일 선박 스트로크의 모델

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

rose-bengal-photothrombosis-confocal-optical-imaging-vivo-model

Research

JoVE Journal

Medicine

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

15.0K 조회수.  The University of Texas Health Science Center San Antonio. Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection. 

비디오: Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

MicroCT를 사용하여 대비 향상된 베셀 영상

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

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 ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

형광 공 초점 Videomicroscopy를 사용하여 종양 혈관의 생체 내 이미징

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

지주막 하 출혈의 생쥐 모델

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

형광 SAPC-DOPS Nanovesicles을 사용하여 뇌 종양 및 관절염의 생체 광학 이미징

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

같은 뇌 Microslices의 흥분 독성 자극<em&gt; 체외</em&gt; 스트로크의 모델

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

결합<em&gt; 생체</em&gt; 광학 및 μCT 이미징 마우스의 정형 외과 임플란트 감염에 감염, 염증 및 뼈 해부학을 모니터링

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

화학 요법에 의한 혈관 독성 - 실시간<em&gt; 생체 내</em&gt; 선박 손상의 영상

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

쥐의 척수 손상의 모델로 초점 국소 빈혈을 Photothrombosis는 유도

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

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 ...

공 초점 광학 이미징에 의해 로즈 벵갈 Photothrombosis

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