저자는 동물 보호와 그들의 도움을 쇼나 Rideout-Gros, 그리고 알렉 산드라의 Garces 감사드립니다, 그리고 그녀의 기술 지원 핑 우 것이다. 이 작품에 대한 지원 보조금없이 아래의 국립 보건원에 의해 제공되었다. EB003268, 그리고 캐나다 연구 의자 프로그램.

1. 초음파 및 MRI 설치 MRI 호환 세 축 집중 초음파 시스템은 (FUS 인 스트 루먼트 주식 회사, 토론토, 온타리오, 캐나다) 본 연구에서 사용되었다. 두 개의 서로 다른 초음파 트랜스 듀서가 사용되었습니다 통합과 내부 건설 551.5 kHz에서 이리저리 중심 변환기는 (곡률 반경 = 60mm, 외부 직경 = 75mm, 내경 = 20 ㎜)과 1.503 MHz의, 8 섹터 배열 PZT 수중 처음기 (Imasonic 주식 Voray-SUR-L&#39;Orgnon, 프랑스)는 단일 요소 이리저리 초점 변환기 (= 0.8, 조리개는 = 10 센티미터 FN)로 구동. MRI 호환 PVDF 수신기 8 551.5 kHz에서 변환기가 사용되었을 때 음향 방출을 기록하는 데 사용되었다. 서로 다른 장비를 사용하는 경우, 다음은 제안된다 : <ol><li> 범위는 0.25 MHz의 -1.5 MHz 단위 중심 주파수 1 이하의 F-번호 (곡률 / 조리개 반경)와 적절한 보정 초음파 변환기를 선택합니다. </li><li> 물 목욕 Fi를 놓으십시오1.5 T 3 T MRI의 침대에 따뜻하고 degassed, 드 이온화 물로 카이로. 물 목욕은 동물을 보유할 수있는 최고의 플레이트가 있어야합니다. 수면에 직면하고, MRI 호환 세 축 위치 결정 스테이지 또는 시스템 9 일 탱크에 초음파 변환기를 탑재합니다. </li><li> 침투 패널을 통해 초음파 구동 장비 듀서 케이블을 실행합니다. </li><li> 함수 발생기와 전력 증폭기를 사용하여 변환기의 구동 신호를 생성합니다. 반사 전력을 최소화하기 위해 외부 매칭 회로를 사용하십시오. </li><li> 변환기 초점 거리 알면, 수면에서 변환기 포커스를 놓고 보이는 물 분수를 생성하기 위해 물 표면을 sonicate. 단계 1.6 1.7은 생성된 물 분수를 사용하여 포커스를 등록하는 방법에 대해 설명합니다. MRI와 초점에서 젤 및 이미징 인한 온도 상승을 흡수 열을 sonicating 포함 초점을 찾기위한보다 정확한 방법은, 9에서 찾을 수 있습니다 &lt;/&gt;이 저녁을 먹다. </li><li> MR 영상에서 볼 수 있습니다 마커를 사용하여 초점의 위치를​​ 표시한다. 이것은 초점 위에 위치했을 때 물로 채울 것입 중앙 구멍과 접시를 사용하여 수행할 수 있습니다. 변환기의 축 방향으로 좌표가 수면에서 결정할 수있는 동안 물이 채워진 구멍이 MRI에 표시되며, 두 비행기에 초점 좌표를 제공할 것입니다. </li><li> , 3 비행기 localizer 시퀀스를 사용하여 이미지 초점 마커를하고 MRI 좌표 시스템의 초점의 위치를​​ 기록합니다. </li><li> 음향 방출을 모니터링하려면 변환기 초점 겨냥한 물을 욕조에 수동 캐비테이션 검출기 (PCD)로 사용되는 MR-호환 수중 처음기의 8을 장착, 또는 통합 수중 처음기와 변환기를 사용합니다. </li><li> 2 Hz에서 (예 : ATS460, AlazarTech, Pointe-클레어, 퀘벡, 캐나다)로 PRFs 10 MS 최대의 파열을 캡처할 충분히 빠르고 범위 카드로 PCD 케이블을 실행합니다. 케이블은 pene에 접지되어야합니다tration 패널 또는 RF 차폐는 노이즈를 최소화합니다. </li></ol> 2. 동물 준비 <ol><li> isofluorane 가스를 사용하여 동물을 마취. isofluorane는 BBB 장애 10 효과가 있기 때문에 동물 실험이 시작하기 전에 가스를 10 분 정도 날아 갔다고한다. 탈수 또는 기타 부상에서 각막을 보호하기 위해 마취의 시작 부분에서 각 눈을 플레이스 안구 윤활제 연고. </li><li> 전기 면도기를 사용하면 동물의 머리와 목 위로부터 모피를 면도 후 depilatory 크림 (예 : 나이르, 교회 &amp; 드와이트 (주) 프린스턴, 뉴저지, 미국)를 사용하여 남은 모피를 제거하고 순한 비누로 두피를 씻어 물. </li><li> bridine 세차 다음 betadine 피부 스크럽으로 꼬리를 준비합니다. 꼬리 정맥을 시각화하기 위해 알코올로 최종 wipedown을 수행합니다. </li><li> 꼬리 정맥에 3 방향 꼭지와 22 게이지 카테터를 삽입하고 응고 형성을 방지하기 위해 헤파린 / 식염수 혼합물 (33 U / ML)로 플러시카테터 인치 </li><li> 근육내 주사를 통해 주사용 anesthetics을 (40-50 밀리그램 / kg 케타민을, 10 밀리그램 / kg xylazine) 전달하고 isofluorane에서 동물을 제거합니다. </li><li> 상단 플레이트 (Fig.1)의 구멍을 통해서 waterbath 연락 머리의 맨과 함께 초음파 포지셔닝 시스템의 anesthetized 동물 부정사를 놓습니다. 초음파의 작은 금액은 어쩌면 덫을 airbubbles의 기회를 최소화하기 위해 동물의 머리의 상단에 적용 젤. </li><li> 포지셔닝 시스템에 테이프 다리. 머리는 어느 물린 막대를 사용 장소에서 개최, 가능한 경우, 또는 테이프 턱에 걸쳐 확고하게 게재될 수 있습니다. </li><li> 수건과 몸을 따뜻하게 유지하기 위해 물 담요를 순환과 동물을 커버. </li></ol> 3. 대상 선정 <ol><li> 기준 축 T 두뇌의 2 가중과 T 1-가중 MR 이미지를 취득. 1.5 T SC 적합 머리 크기의 RF 수신 표면 코일 MRI 및 전용을 사용하는 경우매개 변수가있을 수 있습니다 : T이 가중 : FSE, TE = 60 MS, TR = 2000 MS 가중 T 1 : FSE, TE = 10 MS, TR은 = 500 MS </li><li> T에서 대상 선택 2-가중 스캔, 심실과 뇌의 중간선을 피하고, 그리고 중간 뇌 깊이를 선택합니다. </li><li> 대상 위치에 변환기 포커스를 이동합니다. </li></ol> 4. Microbubble 준비 Definity의 microbubbles은 (Lantheus 의료 이미징, MA, 미국) BBBD 2,5,7을 유도 microbubble 중재의 FUS 여러 그룹에 의해 사용됩니다. 다른 microbubble 유형에 대한 적절한 복용량은 문학 11,12에서 찾을 수 있습니다. <ol><li> Definity의 microbubbles을 활성화 천천히 18 게이지 바늘을 사용하여 1 ML에 주사기로 작은 볼륨을 그립니다. </li><li> 부드럽게 앞뒤로 플런저를 이동하여 주사기의 갇힌 공기를 제거합니다. 이것은 거품을 깰 수있는 주사기를 준거하지 마십시오. </li><li> 정상의 Definity를 희석<html> 천천히 식염수의 주사기로 Definity의 필요한 볼륨을 주입하여 Definty에 10시 1분 염분의 비율로 염분. 주입 배달이 사용되는 경우, Definity는 50:1 또는 100:1로 더 희석 될 수 있습니다. </li><li> 부드럽게도 외관이 달성될 때까지 철저하게 Definity과 식염수를 섞어 주사기로 바꾸란. 거품이 정지 중에 부유하기 시작했을 경우 즉시 사전 주입으로 주사기의 잔잔한 반전도 필요할 수 있습니다. </li><li> 0.02 ML / Definity의 kg 또는 0.2 ML / 솔루션 kg (10시 1분 희석시)에 근거하여 필요한 복용량 볼륨을 계산합니다. </li></ol> 5. 초음파 배달 <ol><li> 낮은 듀티 사이클 파열이 아닌 지속적인 웨이브 sonications를 사용하여 sonication 매개 변수를 설정합니다. 0.5 MHz로 적절한 sonication 매개 변수 원위치 압력 0.23의 MPA, 1 Hz에서 PRF, 2 분 10 MS의 파열입니다. 총 sonication 시간. 현장에서 압력에 적합한 1.5 MHz의는 0.45-0.5 MPA 주위에 빠지다. </li></ol>_content는 &quot;서로 다른 주파수에서&gt; 적절한 압력은 0.46 13 기계 인덱스를 사용하여 추정 할 수 있습니다. <ol start="2"><li> 동물의 머리는 여전히 물이 결합되어 있는지 확인합니다. </li><li> 꼬리 정맥 카테터로 천천히 microbubbles을 주사하고 0.5 ML 정상 식염수에 플러시. 분사의 시작과 동시에 sonication을 시작합니다. </li></ol> 6. 치료 결과의 MRI 평가 <ol><li> sonication 후, 0.5 ML 식염수 수세식이어서 꼬리 정맥 카테터를 통해 (예를 들어 0.2 ML / kg Omniscan, GE 헬스케어) MRI 대비 에이전트를 주입. </li><li> 콘트라스트 피크가 통과하기 전까지는 T 1-가중 영상을 수행합니다. 성공적으로 중단되었습니다 Sonication 사이트는 주변 조직보다 더 큰 향상을 보여줍니다. </li><li> 부종를 확인하기 위해 T 2-가중 영상을 수행합니다. sonication 사이트에서 높은 신호는 부종을 나타내는 것입니다. </li></ol> 7. 대리인결과 MRI 대비 에이전트가 성공적으로 집중 초음파를 사용하고 microbubbles을 순환 BBB를 통해 전달할 수 있습니다. 그림 2는 전형적인 사전 및 사후 FUS T 1-W 이미지를 보여줍니다. 그림 2B는 콘트라스트 향상 (CE) T 1-W의 네 sonicated 지역에서 뚜렷한 초점 구멍과 이미지를 보여줍니다. Sonication 위치 1과 2는 특히 밝은 향상을 보여줍니다. Fig.3 지역의 1과 2는 또한 부종을 나타내는, T 2-w 높은 신호에 대응하는 모습을 볼 수 있습니다. T의 범위 2-W의 부종은 때로 화살 조각에보다 쉽게 시각이 될 수 있습니다. 그림 4는 CE-T 1-W 및 T 2-w 화살 조각 sonication 위치를 통해 1과 3을 보여줍니다. 부종은 지역 하나지만 위치에 3 볼 수 있습니다. 캡처한 음향 방출 데이터 (Fig.5)의 스펙트럼 분석 고조파 방출 및 / 또는 안정 캐비테이션이 발생 초소형 / 서브 고조파 방출을 보여줄 수 있습니다. 하몬서브 및 ultraharmonic 배출량에만 거품 활동 14 결과로 발생할 수 있지만 ICS는 또한, 조직 비 linearities에서 발생할 수 있습니다. 높은 압력에서 관성 캐비테이션을 나타내는 광대역 배출량도 검색됩니다. 그러나 이들은 관성 캐비테이션 11없이 sonications보다 적혈구의 extravasations 및 microdamage보다 양의와 관련된되었습니다. 작은 초점 스폿 크기로 인해 더 많은 지역화된 개구부 높은 sonication 주파수 결과의 사용. 그림 6은 높은 주파수는 오프닝의 작은 영역을 만드는 데 사용할 수있는 것을 보여줍니다. 이것은 적은 가까운 두개골 효과와 효과 중반 두뇌의 조사를 허용합니다. <img alt="1 그림." src="/files/ftp_upload/3555/3555fig1.jpg" /> 그림 1. 실험 설정. <sp클래스 = &quot;pdflinebreak&quot;&gt; <img alt="그림 2." src="/files/ftp_upload/3555/3555fig2.jpg" /> 그림 2. 사전 (왼쪽) 및 사후 (오른쪽) 치료 T 1-W 사 sonication 위치에서 향상을 보여주는 쥐 두뇌의 이미지. <img alt="그림 3." src="/files/ftp_upload/3555/3555fig3.jpg" /> 그림 3. 사전 (왼쪽) 및 사후 (오른쪽) 치료 T 2 - 목 sonication 위치 1과 2에서 2-W의 부종을 보여주는 쥐의 뇌 (Fig.2와 같은 동물)의 이미지. <img alt="4 그림." src="/files/ftp_upload/3555/3555fig4.jpg" /> 그림 4. 우편 치료 화살 T 1-W (왼쪽)과 T 2-W (오른쪽) 같은 쥐의 두뇌에서 이미지는 무화과. 2와 3. 위치 1의 자리 (왼쪽) T 2-w 부종 (오른쪽)와 연결합니다. 위치 3 오프닝 (왼쪽) 그런데 T 2-w 부종을 보여줍니다. </P&gt; <img alt="그림 5." src="/files/ftp_upload/3555/3555fig5.jpg" /> 그림 5. 551.5 kHz에서에서 단일 열 MS 버스트 동안 캡처된 데이터의 주파수 스펙트럼. 기본 진동수 (F 0)뿐만 아니라 고조파 (2 F 0)와 하위 / ultraharmonics은 (0.5 F 0, 1.5 F 0) 볼 수 있습니다. <img alt="6 그림." src="/files/ftp_upload/3555/3555fig6.jpg" /> 그림 6. 후 치료 CE-T 1-W 축 (왼쪽)와 1.503 MHz로 네개의 장소에 sonicated 쥐 두뇌의 화살 (오른쪽) 이미지. 이 주파수에서 BBB 채용 더화된 것으로 볼 수 있습니다.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+&#40;BBB&#41;+disruption+using+a+diagnostic+ultrasound+scanner+and+Definity+in+mice.">Blood-brain barrier &#40;BBB&#41; disruption using a diagnostic ultrasound scanner and Definity in mice.</a> Ultrasound Med. Biol. 35, 1298-1308 (2009).</li><li>O'Reilly, M. A., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PVDF+receiver+for+ultrasound+monitoring+of+transcranial+focused+ultrasound+therapy.">A PVDF receiver for ultrasound monitoring of transcranial focused ultrasound therapy.</a> IEEE Trans. Biomed. Eng. 57, 2286-2294 (2010).</li><li>Chopra, R., Curiel, L., Staruch, R., Morrison, L., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+MRI-compatible+system+for+focused+ultrasound+experiments+in+small+animal+models.">An MRI-compatible system for focused ultrasound experiments in small animal models.</a> Med. Phys. 36, 1867-1874 (2009).</li><li>McDannold, N., Zhang, Y., Vykhodtseva, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-Brain+Barrier+Disruption+and+Vascular+Damage+Induced+by+Ultrasound+Bursts+Combined+with+Microbubbles+can+be+Influenced+by+Choice+of+Anesthesia+Protocol.">Blood-Brain Barrier Disruption and Vascular Damage Induced by Ultrasound Bursts Combined with Microbubbles can be Influenced by Choice of Anesthesia Protocol.</a> Ultrasound Med. Biol. , (2011).</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+blood-brain+barrier+with+focused+ultrasound:+association+with+cavitation+activity.">Targeted disruption of the blood-brain barrier with focused ultrasound: association with cavitation activity.</a> Phys. Med. Biol. 51, 793-807 (2006).</li><li>Yang, F. -. Y., Liu, S. -. H., Ho, F. -. M., Chang, C. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ultrasound+contrast+agent+dose+on+the+duration+of+focused-ultrasound-induced+blood-brain+barrier+disruption.">Effect of ultrasound contrast agent dose on the duration of focused-ultrasound-induced blood-brain barrier disruption.</a> J. Acoust. Soc. Am. 126, 3344-3349 (2009).</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+induced+by+focused+ultrasound+and+circulating+preformed+microbubbles+appears+to+be+characterized+by+the+mechanical+index.">Blood-brain barrier disruption induced by focused ultrasound and circulating preformed microbubbles appears to be characterized by the mechanical index.</a> Ultrasound Med. Biol. 34, 834-840 (2008).</li><li>Neppiras, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+Cavitation.">Acoustic Cavitation.</a> Physics Reports (Review Section of Physics Letters). 61, 159-251 (1980).</li><li>O&#39;Reilly, M. A., Huang, Y., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+standing+wave+effects+on+transcranial+focused+ultrasound+disruption+of+the+blood-brain+barrier+in+a+rat+model.">The impact of standing wave effects on transcranial focused ultrasound disruption of the blood-brain barrier in a rat model.</a> Phys. Med. Biol. 55, 5251-5267 (2010).</li></ol>

K. Hynynen과 R. 초프라은 FUS 인 스트 루먼트의 공동 설립자이며, 주식 회사 R. 초프라, A. Waspe 및 K. Hynynen는 주주 FUS 인 스트 루먼트에 주식 K. Hynynen은 FUS 악기 주식 회사로부터 연구 지원을받는

동물과 microbubbles의 준비는이 절차의 가장 중요한 측면입니다. 동물의 머리에 머리를 완전히 초음파 빔을 attenuating 피하기 위해 제거해야합니다. BBB는 isofluorane 마취하에 중단될 수 있습니다 그러나, 그것은 일관된 개방을 달성하기 위해 더 어려워집니다. 그들을 위반하지 않도록하기 위해, 최대 그릴 때 microbubbles 항상 관심과 작은 게이지해서 다루어 져야, 큰 직경의 바늘을 ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 작은 동물들은 초음파 시스템을 중점 </td><td> FUS 인 스트 루먼트 주식 회사 </td><td> RK-100 </td></tr><tr><td> Definity </td><td> Lantheus 의료 이미징 </td><td></td></tr><tr><td> Omniscan </td><td> GE 헬스케어 </td><td></td></tr></tbody></table>

mri-guided disruption of the blood-brain barrier using transcranial focused ultrasound in a rat model

혈액 - 뇌 장벽 (BBB)의 초점 초음파 (FUS) 혼란은 BBB 1-5 우회 위해 점점 더 조사 기법입니다. 덜 크기가 6에서 약 500 다보다 분자로 뇌 조직에 vasculature에서 분자의 통과 제한으로 BBB는 뇌 장애의 제약 치료에 중요한 장애물이다. FUS 유도 BBB 장애 (BBBD)는 화학 물질 이상의 장점은 고도화된 됨으로써 BBBD 일으킬 의미 임시 및 4 가역하고 있습니다. FUS 유도 BBBD 달리 충분한 농도의 조직에 deliverable 않을 두뇌에 치료 요원들이 다양한 효과를 조사하는 수단을 제공합니다. 초음파 파라미터의 다양한 범위 BBB 2,5,7을 방해 성공적으로 증명했지만, 정확한 타겟팅과 성공적인 중단을 보장하기위한 실험 절차의 몇 가지 중요한 단계가 있습니다. 이 protoc좋았 윤곽선은 얼마나 중요한 동물의 준비와 실험의 microbubble 처리 단계를 중심으로 쥐 모델에서 BBBD 유도된 MRI-안내 FUS을 달성합니다.

Watch this Scientific Journal Video about MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model at JoVE.com

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

혈액 - 뇌 장벽 (BBB)​​의 Microbubble-매개 집중 초음파 장애는 두뇌의 비침습 타겟 약물 전달을위한 유망 기술입니다<sup&gt; 1-3</sup&gt;. 이 프로토콜은 쥐 모델에서 MRI를 유도 transcranial BBB 장애에 대한 실험 절차를 설명합니다.

쥐 모델에서 Transcranial 집중 초음파를 사용하여 혈액 - 뇌 장벽의 MRI를 유도 장애

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

mri-guided-disruption-blood-brain-barrier-using-transcranial-focused

Research

JoVE Journal

Medicine

17.8K 조회수.  Sunnybrook Research Institute. 혈액 - 뇌 장벽 (BBB)​​의 Microbubble-매개 집중 초음파 장애는 두뇌의 비침습 타겟 약물 전달을위한 유망 기술입니다 1-3. 이 프로토콜은 쥐 모델에서 MRI를 유도 transcranial BBB 장애에 대한 실험 절차를 설명합니다.

비디오: MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

자기 공명 영상, 체적 계산의 Tomography 및 초음파를 이용한 유방암의 뼈 전이의 누드 쥐 모델에서 Angiogenesis의 멀티 모달 이미징

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

연구

의학

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

혈액 - 뇌 장벽 장애의 쥐 모델 Neurovascular 치료학 타겟이 분명한 허용하려면

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

쥐의 만성 영양 주입의 모델

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

로 뇌 미세 혈관 내피 세포 (CEnd를)에 기지개<em&gt; 체외</em혈액 뇌 장벽의&gt; 외상성 뇌 손상 모델

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

설정 - 업<em&gt; 체외</em쥐 뇌 혈관 벽의&gt; 모델 (BBB)​​ : BBB 불 침투성 및 수용체 매개 운송에 초점

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

오름차순 대동맥 강성 측정<em&gt; 생체</em&gt; 초음파를 사용하여 마우스의

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

같은 산소 포도당 박탈과 재산 소화<em&gt; 체외</em&gt; 허혈 - 재관류 손상 모델 공부 뇌 혈관 장벽 부전

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

치료 저항성 주요 우울 장애에 대한 치료로 dmPFC-RTMS를 자기 공명 영상 유도

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

혈액 뇌 장벽 메쉬와 유사한 혈관 형성 및 분열을 평가하기위한 In vitro 시험 법

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...