S 엉터리 Drottning Viktorias Frimurarstiftelse :이 작품은 Karolinska Institutet 아이에게 자금, 스웨덴어 방사선 보호 기관, Karolinska Institutet 안구 연구 재단, 총 엉터리 Bertil Stohnes Stiftelse, 세인트 에릭의 눈 병원 연구 재단, Ögonfonden, Konung 구스타프 V에 의해 지원되었다.

1. 자외선 방사선에 노출 <ol><li> 15 분 노출되기 전에, 90 밀리그램 / kg Ketalar (케타민)와 intraperitoneal 주사로 10 밀리그램 / kg Rompun (xylazine)의 혼합물 여성 Sprague-Dawley 쥐를 마취. </li><li> 쥐 restrainer에서 동물을 놓고 트렁크 짜 13 발생하지 않고 쥐의 고정까지 벨트를 조이십시오. </li><li> mydriasis를 유도 할 수있는 쥐의 두 눈에 Mydriacyl (tropicamide), 10 MG / ML을 심어. </li><li> 그 한쪽 눈이 절반이 최대 10 nm의 전체 폭이 300 nm의 14 UVR의 좁은 빔에 대해 위치 될 수 있도록 동물을 놓고 검은 색 테이프 contralateral 눈을 보호. </li><li> 15 300 nm의 분 15에서 쥐 일방적으로 하위 임계 복용 한 kJ / m 2 UVR-B을 쉽게받을 수 있습니다. </li></ol> 2. 해부 <ol><li> 미리 정해진 후 노출 간격 (1, 5, 24 및 120 시간) 후, carbo하여 육주 늙은 쥐 (체중 &lt;200g)를 희생N 개의 이산화 질식과 경추의 전위. </li><li> 명백히하다 눈. 이후, 아래 뒤쪽면을 눈 각막을 넣어. 그런 다음, 펀치 27 게이지 캐뉼라를 적용하고 공막에서 매우 가깝습니다 렌즈 손상을 방지하기 위해 공막에 tangentially 밀어로 최소한의 구멍. 그런 다음 공막의 뒤쪽 부분을 들어 올려 수있을 때까지 그냥 limbus 뒤에 원주 잘라 안과 수술 가위를 사용합니다. 그런 다음, 무딘 곡선 포셉으로 렌즈를 올린다. </li><li> 5-10 분보다 더 이상 균형 소금 솔루션에 렌즈를 유지, 현미경 렌즈 적도에서 모양체의 잔해를 제거합니다. </li></ol> 3. 정량 RT-PCR <ol><li> 350 μl RA1 용해 완충액 (NucleoSpin RNA II 총 RNA 분리 키트)와 2 ML Eppendorf 튜브에 3.5 μl β-메르 캅토 에탄올의 혼합에 렌즈를 삽입합니다. </li><li> 실온에서 30 분 동안이 혼합에 렌즈를 보관. </li><li> 로 렌즈를 균질화렌즈 만 하드 핵까지 바늘이 렌즈 (피질과 렌즈의 캡슐이 용해 버퍼에 lysed 아르)에서 남아있다. 믹스에서 핵을 제거합니다. </li><li> 스토어 샘플 즉시 -70시 ° C. </li><li> 샘플을 해동하고 프로토콜 &quot;교양 세포와 조직의 총 RNA 정화&quot;(NucleoSpin RNA II 총 RNA 분리 키트)를 따르십시오. </li><li> 스토어 RNA 샘플 -70에서 ° C. </li><li> 2 분 95 ° C, 40 사이클에 대한 2 단계 : 각 샘플에서 DNA의 충분한 제거를 확인하려면 다음과 같은 조건 (1 단계에서 PCR을 실행 95 ° C 20 초, 55 ° C 20 초에 72 ° 20 초에 C, 3 단계 : 7 분 72 ° C) p53 유전자의 DNA 특정 프리 머와 함께 앞으로 5&#39;-ACCCTCTGACCTTTTTCCCA-3 &#39;와 역 5&#39;-TGCTGGGATCTTAGGCACTC-3&#39;와 DNA 형성 촉매의 DNA 중합 효소 (dNTPack), 제조에 따라 프로토콜입니다. 예상 PCR 제품은 243 기본 쌍입니다. </li><li> 243 기본 쌍 DNA 특정 PCR 제품을 감지 1.5 % 아가로 오스 겔 전기 영동을 실행합니다. 준비하기TBE 버퍼에 1.5 % 아가로 오스 겔은 아가로 오스의 3.75 g을 가지고 TBE 버퍼 250 ML에 해결. ethidium 브로마이드 (0.5 MG / ML) 1.5 % 아가로 오스 겔 솔루션 500 ML 500 μl를 추가합니다. 전자 레인지 오웬의 혼합물을 가열하고 아가로 오스를 해결하기 위해 젓는다. </li></ol> 샘플 중 어느 것도 1.5 % 아가로 오스 겔 전기 영동에있는 모든 DNA 특정 PCR 제품을 나타 내기 위해이 없습니다. <ol start="9"><li> RNA 샘플 (1 μl)의 농도와 NanoDrop ND-1000 분광 광도계에서 260 나노 미터 (RNA)와 280 nm의 (단백질)에서 샘플 흡광도의 비율을 측정합니다. RNA 샘플 흡광도의 단백질에 대한 비율 RNA 2.0 이상이면 다음 RNA 샘플은 순수합니다. RNA 샘플 흡광도의 비율이 2.0보다 낮 으면 정화 단계 3.5 RNA 다시 실행. </li><li> 1 μg에 해당하는 RNA 샘플의 볼륨을 타고 프로토콜 1의 스트랜드 cDNA 합성 키트 (RT-PCR을위한 1 차 스트랜드 cDNA 합성 키트)에 따라 cDNA 합성. </li><li> -20 ° C에서 cDNA 샘플을 저장(기간 1과 년 이상, -70에서 저장 ° C). </li><li> iCycler MyiQ 단일 색상 실시간 PCR 검출 시스템에 정량 실시간 PCR을 실행합니다. 로드는 1 μl cDNA 샘플, TaqMan 유전자 표현 마스터 믹스, caspase 3 TaqMan 유전자 발현 분석과 함께 96 - 웰 플레이트에 triplicates 지침을 제조했다. 로드는 1 μl cDNA 샘플, TaqMan 유전자 표현 마스터 믹스, 18에 대한 TaqMan 유전자 발현 분석과 또 다른 96 - 웰 플레이트에 triplicates 안내 (TaqMan 유전자 발현 분석 프로토콜)를 제조했다. 시리즈에서 세 무작위로 선택한 비 노출 렌즈의 cDNA의 일부를 희석. 96 - 웰 플레이트에 샘플의 cDNA와 함께 시리얼 dilutions을 실행합니다. </li><li> 결과의 정량화에 대한 표준 곡선 방법을 사용합니다. 임계 형광의 사이클의 수는 MyiQ 소프트웨어의 측정으로 사용됩니다. 캘리브레이터의 상대적 농도의 함수로 입구에서 사이클의 수를 표현하는 표준 곡선은 에스입니다각 플레이트에 시리얼 dilutions을 위해 tablished. 세 이외의 치료 렌즈 샘플의 cDNA는 보정을 위해 사용되었다. cDNA가 측정 샘플의 상대적인 농도를 얻기 위해 표준 곡선에 각 샘플에 대한 사이클의 임계 번호를 관련. 마지막으로, cDNA 내부 제어 18에 cDNA 대상의 상대적 농도를 관련된하여 대상 유전자의 표현 수준을. </li></ol> 4. Immunohistochemical 착색 <ol><li> 정착 <ol><li> PBS의 눈을 (; 산도 7.4 인산 식염 버퍼링) 해부. </li><li> 20 분을위한 얼음처럼 차가워에 4 % paraformaldehyde (PFA)로 가득 찬 튜브에 눈을 놔. 전날 PFA 4 % 준비합니다. 4시 매장 ° C 사용까지. PFA는 약 1 주일 동안 신선한 것입니다. </li><li> micropipette으로 PFA를 제거합니다. 그런 다음 20 분을위한 얼음 차가운 PBS로 씻어. </li><li> ° C 밤 동안 4에서 자당 30 %가 눈을 놔. </li><li> 최적의 절삭 온도 (OCT) 매체로 가득 찬 컵에 눈을 넣어 (조직- 테크). sectioning에 적합한 위치에 눈을 놔. </li><li> 드라이 아이스를 통해 월 중간에 멈춰 봐. </li><li> -70에서 저장 ° C 사용까지. </li></ol></li><li> Sectioning <ol><li> 해당 비행기에 cryo-sectioning에 눈을 배치합니다. </li><li> 각 렌즈의 중앙 부분에서 세 5 μm 두께의 중간 시상 부분을 잘라 버릴거야. 다른 섹션에 동일한 핵을 얼룩 방지하기 위해 연속 섹션 사이에 최소 6 절을 폐기하십시오. </li><li> 섹션 절단되면 부드럽게 상단에 슬라이드를 놓습니다. 섹션은 다음 슬라이드에 충실해야합니다. </li><li> 사용하기 전에 공기 건조에 슬라이드를 둡니다. 필요 할 때까지 슬라이드는 -20 ° C에서 저장 될 수 있습니다. </li></ol></li><li> 형광 immunohistochemistry <ol><li> 샘플 실온 (5-10 분)을 달성 보자. </li><li> PAP-펜 (Invitrogen)를 표본 주위에 가장자리를 그립니다. </li><li> 잠시 (10 분)에 테두리가 건조 보자. </li><li> 현미경 슬라이드를 붙입니다. </li><li> 1 Rehydrate × P를15 분을위한 BS. </li><li> 30 분에 표준 블록 솔루션 Permeabilize. </li><li> 표준 블록 솔루션에 1:3,000를 희석 (세포 Signalling 기술, Inc의 토끼 polyclonal, 흘리고 caspase-3 항체 Asp175 966​​1)가 기본 항체를 추가합니다. </li><li> 밤 동안 4 ° C에서 humidified 챔버에 저장합니다. </li><li> (3 × 5 분) pipetting 또는 대형 욕조 (&gt; 15 분, 1-2 변경)에 immersing하여 PBS로 씻으십시오. </li><li> 표준 블록 솔루션에 1:300을 희석 보조 항체를 (특정 흡수 / 방출 스펙트럼과 안티 토끼 차 항체)를 추가합니다. </li><li> (3 × 5 분) pipetting 또는 대형 욕조 (&gt; 15 분, 1-2 변경)에 immersing하여 PBS로 씻으십시오. </li><li> PAP-펜 얼룩을 닦아. </li><li> 3 시간 동안 실온에서 humidified 챔버에 저장합니다. 빛에 노출을 피하십시오. </li><li> Vectashield 한 방울을 추가하고 슬라이드 커버 슬립을 배치합니다. 거품을 피하십시오. </li><li> Vectashield 굳어를 (~ 20 분) 보자. </li><li> 섹션 바로 내가 보관분석까지 어둠 습니. </li><li> 모든 제어 섹션은 기본 항체의 부재로 처리됩니다. </li><li> 형광 현미경으로 몇 시간 안에 결과를보세요. </li><li> 한쪽 핵 활에서 각 렌즈의 반대편 핵 활에 상피 세포 &#39;핵을 계산합니다. 파란색의 모든 렌즈 상피 핵 녹색의 caspase-3 긍정적 핵을 보려면 차 항체의 방출에 맞는 표준 필터를 확인하기 위해 표준 청색 필터를 적용 할 수 있습니다. </li><li> 모든 렌즈 상피 핵 및 긍정적 인 핵의 수를 기록합니다. 각 섹션에 대한 세포 세 번 계산합니다. </li></ol></li></ol>

<ol>
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G., 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=Toxicity+of+ultraviolet+radiation+exposure+to+the+lens+expressed+by+maximum+tolerable+dose+(MTD).">Toxicity of ultraviolet radiation exposure to the lens expressed by maximum tolerable dose (MTD).</a> Developments in Ophthalmology. 35, 70-75 (2002).</li><li>Michael, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+repair+of+cataract+induced+by+ultraviolet+radiation.">Development and repair of cataract induced by ultraviolet radiation.</a> Ophthalmic Research. 32, 1-45 (2000).</li><li>L&#246;fgren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cataract from ultraviolet radiation. , (2001).</li><li>Ayala, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Influence of exposure patterns and oxidation in UVR induced Cataract. , (2005).</li><li>Dong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Safety limit estimation for cataract induced by ultraviolet radiation. , (2005).</li><li>L&#246;fgren, S., Michael, R., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+age+and+sex+in+ultraviolet+radiation+cataract+in+the+rat.">Impact of age and sex in ultraviolet radiation cataract in the rat.</a> Investigative Ophthalmology and Visual Science. 44, 1629-1633 (2003).</li><li>Ayala, M. N., Michael, R., S&#246;derberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+exposure+time+for+UV+radiation-induced+cataract.">Influence of exposure time for UV radiation-induced cataract.</a> Investigative Ophthalmology and Visual Science. 41, 3539-3543 (2000).</li></ol>

관심 없음 충돌이 선언 없습니다.

<html> 본 논문은 UVR-B 유발 백내장 동안 발생하는 분자 이벤트를 공부하고 사용 방법을 설명합니다. 생체 UVR 유도 백내장에서 사용할 수 대부분의 정보는 흰둥이 Sprague-Dawley 쥐 7, 16, 17, 18, ​​19에서 실험에서 파생 된 것으로, 고려, 우리는 현재의 연구에 흰둥이 Sprague-Dawley 쥐를 사용하기로 결정했습니다. 쥐의 나이 여섯 일주일 정도했습니다. 남성과 대?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> Ketalar </td><td> 화이자 </td><td> 150086 </td><td> 50 MG / ML </td></tr><tr><td> Rompun </td><td> 바이엘 </td><td> 022545 </td><td> 20 MG / ML </td></tr><tr><td> Oculentum 단순 </td><td> Apoteket, 스웨덴 </td><td> 336164 </td><td> 5g </td></tr><tr><td> Mydriacyl </td><td> 앨콘 </td><td> 00,352 </td><td> 10 MG / ML </td></tr><tr><td> 균형 소금 솔루션 </td><td> 앨콘 </td><td> 0007950055 </td><td></td></tr><tr><td> 3.5 UL β-메르 캅토 에탄올 </td><td></td><td></td><td></td></tr><tr><td> NucleoSpin RNA II 총 RNA 분리 키트 </td><td> Macherey-Nagel GmbH의 및 공동, Duren, 독일 </td><td> 740955.50 </td><td></td></tr><tr><td> p53의 DNA의 특정 프리 머 </td><td> biomers.net GmbH의 </td><td> 사용자가 만든 </td><td></td></tr><tr><td> DNA 형성 촉매의 DNA 중합 효소, dNTPack </td><td> 로슈 </td><td> 04 728 866 001 </td><td></td></tr><tr><td> 아가로 오스 </td><td> 시그마 </td><td> A5093 </td><td></td></tr><tr><td> Ethidium 브로마이드 솔루션 0.5 MG / ML </td><td> 시그마 </td><td> E1385 </td><td></td></tr><tr><td> 나노 드롭 ND-1000 분광 광도계 </td><td> NanoDrop 제품 </td><td></td><td></td></tr><tr><td> RT-PCR을위한 1 차 스트랜드 cDNA 합성 키트 (AMV) </td><td> 로슈 진단 GmbH의 </td><td> 11 483 188 001 </td><td></td></tr><tr><td> iCycler MyiQ 단일 색상 실시간 PCR 탐지 시스템 </td><td> 바이오 RAD 연구소 </td><td></td><td></td></tr> &lt;TR&gt; <td> 96 - 웰 플레이트 </td><td> Sarstedt </td><td> 72.1979.202 </td><td></td></tr><tr><td> TaqMan 유전자 발현 마스터 믹스 </td><td> 응용 Biosystems </td><td> 4369016 </td><td></td></tr><tr><td> caspase 3 TaqMan 유전자 발현 분석 </td><td> 응용 Biosystems </td><td> Rn00563902_m1 </td><td></td></tr><tr><td> 18에 대한 TaqMan 유전자 발현 분석 </td><td> 응용 Biosystems </td><td> Hs99999901_s1 </td><td></td></tr><tr><td> MyiQ 소프트웨어 </td><td> 바이오 RAD 연구소 </td><td></td><td></td></tr><tr><td> 흘리고 caspase-3 (Asp175) </td><td> 세포 신호 기술 </td><td> 9661 </td><td></td></tr><tr><td> 안티 토끼 IgG </td><td> Abcam </td><td> Ab6798 </td><td></td></tr><tr><td> 자당 </td><td> 시그마 알드리치 </td><td> 84,097 </td><td></td></tR&gt; <tr><td> Vectashield </td><td> 벡터 연구소 </td><td></td><td></td></tr><tr><td> 유니버설 현미경 Axioplan이 영상 </td><td> 칼 Zeiss </td><td></td><td></td></tr><tr><td> Paraformaldehyde </td><td> 시그마 알드리치 </td><td> 441244 </td><td></td></tr><tr><td> 10X TBE 버퍼 </td><td> Promega </td><td> V4251 </td><td></td></tr></tbody></table>

characterization of molecular mechanisms of in vivo uvr induced cataract

백내장은 세계 1 실명의 주요 원인이다. 세계 보건기구 (WHO)는 빛의 전송을 막는 눈의 수정체의 뿌옇게로 백내장 정의합니다. 백내장은 당뇨병, 흡연, 자외선 방사선 (UVR), 알코올, 이온화​​ 방사선, 스테로이드 및 고혈압과 관련된 여러 요소 질병입니다. UVR은 백내장 일으키는 강력한 2-4 실험 및 역학 증거 5,6가 있습니다. 우리는 anesthetized 7 비 anesthetized 동물 팔 모두에 UVR B 유발 백내장에 대한 동물 모델을 개발했습니다. 백내장에 대한 유일한 치료는 수술이지만이 치료는 모든 액세스 할 수 없습니다. 그것은 10 년 동안 백내장의 발병을 지연 50 % 9 백내장 수술에 대한 필요성을 줄일 수있는 것으로 추산되었습니다. 백내장의 발생을 지연하기 위해, 그것은 백내장 형성의 메커니즘을 이해하고 효과적인 예방 strat를 찾을 필요합니다egies. 백내장 개발을위한 메커니즘 중 apoptosis 인간과 동물 10 백내장의 개시에 중요한 역할을 담당하고 있습니다. 우리의 초점은 최근 백내장 개발 8,11,12을위한 메커니즘으로 렌즈의 apoptosis있다. 그것은 apoptosis 경로에 UVR의 효과를 더 잘 이해 백내장 방지 할 수있는 새로운 의약품의 발견에 대한 가능성을 제공 할 것으로 예상된다. 이 문서에서는, 우리는 실험적으로 UVR-B에 생체 노출에 의해 유도 할 수있는 방법 백내장 설명합니다. 또한 RT-PCR 및 immunohistochemistry이 도구 UVR-B 유발 백내장의 분자 메커니즘을 연구하기로 제공됩니다.

측정의 유사 콘텐츠의 다양한 소스는 분산 분석과 예상되었고 그것은 동물 당 3 측정을 고려하면 측정을위한 분산 동물에 대해 해당의 15 %의 순서에 있던 것을 발견했습니다. 따라서, 전체 렌즈 분석을 고려, 그것은 정밀도를 향상 할 수 없습니다. 직교 테스트 120 시간의 대기 시간 대 짧은 지연 시간 간격 사이의 caspase-3 메시지 통계적으로 의미있는 대조를 elucidated. 생체</...

Watch this Scientific Journal Video about Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract at JoVE.com

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

백내장은 세계에서 실명의 주요 원인이다. 태양 자외선 방사 (UVR)는 백내장 개발을위한 주요 위험 요소입니다. 지금까지 UVR-B 유발 백내장의 동물 모델은 개발되었습니다. UVR, 정량 RT-PCR 및 immunohistochemistry에 노출 :이 글에서 우리는 백내장 형성에 대한 조사를하는 방법을 설명합니다.

의 분자 메커니즘의 특성<em&gt; 생체에</em&gt; UVR 유도 백내장

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

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Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

9.8K 조회수.  Karolinska Institutet. 백내장은 세계에서 실명의 주요 원인이다. 태양 자외선 방사 (UVR)는 백내장 개발을위한 주요 위험 요소입니다. 지금까지 UVR-B 유발 백내장의 동물 모델은 개발되었습니다. UVR, 정량 RT-PCR 및 immunohistochemistry에 노출 :이 글에서 우리는 백내장 형성에 대한 조사를하는 방법을 설명합니다.

비디오: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

염증성 플라크의 적외선 형광 (NIRF) Intravascular 분자 이미징, Multimodal 접근 가까운 생체내에서 죽상 경화증의 영상을

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

연구

의학

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

생체내에</em수축 유도 부상 및 복구의 비침습 모니터링> 쥐 모델

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

세포 추적을위한 생체 19 F MRI에서

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

분자 대장 암 세포의 3 차원 모델의 침윤성 종양 미세 환경의 프로파일 및<em&gt; 예 생체 내</em&gt; 섬유 아세포

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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 ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

마우스 모델의 설립 및 요로 감염 및 CAUTI의 특성

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

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 ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

생체 외에서 그리고 Vivo에서 인간 세포, C. 선 충, 쥐에서 Mitophagy의 검출

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

비보에서 확장 된 심근병증의 독소루비신 유도 뮤린 모델

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

마우스에서 우측 관상동맥의 영구적 결찰에 의해 유도되는 우심실 심근경색의 생성 및 특성화

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...