이 작품은 실명 (RPB)을 방지하기 위해 NIH 교부금 1R01EY022901, 연구에서 경력 개발 수상에 의해 지원되었다, CMReeves &amp; MA 리브스 재단, 시각 장애인 E. 마틸다 지글러 재단, 기사단 눈 재단, 그리고학과에 제한없이 부여 RPB 유타 대학의 안과. 우리는 원고에 대한 토론과 의견에 대한 Spectralis 멀티 양상 이미징 시스템 및 타오 장에 기술 지원을 Balamurali Ambati 감사합니다.

1. 악기의 제조 <ol><li> 절차는 동물 시설에서 절차 룸에서 수행된다. </li><li> 실험을 시작하기 전에 안면 마스크, 헤어 보닛, 수술 가운, 멸균 발 덮개, 장갑을 착용 할 것. </li><li> 열판에서 비커에 ~ 40 ° C의 열 물. </li><li> 촬영하는 동안 마우스의 체온을 유지하기 위해 나중에 사용되는 가열 패드 위에 멸균 블루 패드를 놓습니다. 가열 패드의 전원을 켭니다. </li><li> 이미징 시스템 준비 <ol><li> 먼지 덮개를 제거하고 레이저의 전원을 켭니다. </li><li> 55 ° 렌즈를 꺼내 기계에 마운트. </li><li> 컴퓨터 입력 새로운 환자의 시트에서 이미징을위한 마우스의 정보 (예를 들어, 유전자형, 연령, 등.)에서 이미징 소프트웨어를 엽니 다. &quot;디바이스 유형&quot;에서 적외선 (IR​​) 모드를 선택합니다. </li></ol></li></ol> 주 :보고 된 것을 사용 O우리는 (대표적인 결과를 참조 1-4 피규어) 외부 렌즈를 사용하지 않고, IV-ICGA하여 고품질의 화상을 획득하는데 아무런 문제가 없지만 F 외부 이중 비구면 렌즈는 화질 17-20을 향상시킬 수있다. 2. ICGA의 꼬리 정맥 주입 <ol><li> 1 % Tropicamide 안과 솔루션을 마우스 눈을 넓히고 5 분을 기다립니다. </li><li> 필요한 마취 (케타민 / 자일 라진 / 아세 프로 마진 65-100/10-20/1-3 ㎎ / kg)의 양을 결정하기 위하여 마우스를 체중. </li><li> 32 G 바늘과 함께 멸균 된 1 ML의 주사기를 검색합니다. 마취제 (13 ㎎ / ㎖ 케타민, 2.6 ㎎ / ㎖ 자일 라진, 멸균 PBS에 0.​​3 ㎎ / ㎖ 아세 프로 마진)과 복강 마우스를 주입한다. 마우스가 완전히 마취 될 때까지 기다립니다 (~ 5 분). </li><li> 정맥의 혈관 확장을 일으키는 원인이되기 위하여 40 ° C의 따뜻한 물에 마우스 꼬리를 놓습니다. </li><li> 32 G 바늘로 1 ML의 주사기를 검색합니다. IC의 원하는 금액을 인출G, 전형적으로 25g의 마우스 (2 ㎎ / kg)을 위해, 멸균 튜브에 0.2 μM 주사기 필터로 여과 멸균 1 ㎎ / ㎖ ICG, 50 μL. 주사기에있는 공기를 도입하지 않도록주의하십시오. </li><li> 주입 할 영역을 소독하기 위해 알코올 면봉으로 꼬리를 닦아냅니다. </li><li> 측면 꼬리 정맥이 상승되도록 한 손으로 꼬리를 잡고. 향하고 바늘의 베벨, 최소 각도로 정맥 ~ 2mm를 바늘을 주입. 정맥을 관통하지 않도록주의하십시오. 약간 주사기에 다시 그려 바늘이 성공적으로 정맥에 삽입 된 것을 나타내는 바늘 허브로 혈액의 흐름의 흔적을 찾습니다. </li><li> 천천히 정맥에 ICG를 주입. 주입 할 때 최소한의 저항이 있어야합니다. 바늘을 제거하고 출혈을 멈추게하기 위해 ~ ~ 10 초 동안 주사 부위에 직접 알코올 면봉을 적용합니다. 마우스가 촬영을위한 다음 준비가되어 있습니다. 초기 단계 (주입 후 0-4 분)을 잡기 위해서는, ESSE이며신속하게 이미지에 마우스를 ntial. </li></ol> 참고 : 마우스의 눈은 쉽게 건조 얻을 수 있으며 마취 백내장을 개발할 수 있습니다. 이 절차를 수행하는 동안 멸균 PBS를 적용하여 촉촉한 눈을 유지하는 것이 중요합니다. ICGA 녹화 전에 멸균 면봉으로 초과 PBS를 닦아냅니다. 다른 실험실 각막 17-20의 탈수를 방지하기 위해 콘택트 렌즈를 사용했다. 3. ICG 혈관 조영술 <ol><li> 망막 및 맥락막 순환이 최대 밝기 (0-4 분)에 될 때까지 이미지보기 맥락막 작성의 초기 단계의 캡처를 허용 ICG 주입 후 30 ~ 40 초를 가지고 시작합니다. 망막 혈관은 최적의 초점 ~ 35 ~ 45 디옵터 및 맥락막 혈관이 10 ~ 15 디옵터에서 시각화에서 시각화됩니다. 주 : 동물 모델의 첫 번째 시험 동안, 모든 가능한 abno를 식별하기 위해 모든 각도 (코, 시간, 지느러미와 복부)에서 이미지를 캡처하는 것이 좋습니다혈관에 rmalities. 초기 단계 동안, 중형 및 대형 맥락막 동맥과 정맥 모두 잘 시각화된다. 이 프로토콜에 사용되는 동물 모델에서, 맥락막 병변 (예 : 결절 황반부)의 초기 단계에 1 분을 표시 시작할 수 있습니다. </li><li> 혈관에 이미지 초점을 설정합니다. 밝기 제어 및 각각 제어 모듈과 초점 노브를 사용하여 초점을 맞 춥니 다. 이 값은 조정 디지털로 쉽게 일정하게 유지된다. 다음 기술을 이용하여 화질이 재현되도록 ​​상기 카메라 렌즈 상수 마우스의 눈으로부터의 거리를 유지한다. 주 : 디바이스는 이미지, 우리는 카메라 렌즈와 일정한 마우스 눈 간의 포커스, 밝기 및 거리를 유지하는 후방 눈 부 시도 할 수 있기 때문에, 우리는 이미지를 다른 각도에서 전체 후방 눈 등. 이것의 핵심은 VIE의 필드와 눈을 통해 ICG 의해 방출 원 형상의 발광을 정렬하는 것이다카메라의 승. 전체 이미지가 더 어두운 영역이 없을 때까지이 작업은, 왼쪽에서 오른쪽으로 만들어 카메라 위치의 상하 및 안팎으로 조정을 수행 할 수 있습니다. 카메라의 뷰의 발광 및 필드가 줄 때, 렌즈에 눈의 거리가 품질의 영상을위한 최적의 거리에서 다음 이미지의 설정뿐 아니라 재현 할 것입니다. </li><li> 맥관 구조에 초점이 맞춰지면, 획득 모듈에 둥근 검은 버튼을 누르면 이미지 프레임을 캡처. 라운드 블랙 버튼은 최고의 화질 ICG의 신호를 감소 시키거나 향상시키기 위해 사용될 수있다. </li><li> 이미지 맥락막 병변에 최적의 화각과 초점 깊이를 결정합니다. 그것은 눈의 위치, 초점 심도, 및 전체 시간 코스 ICGA 고정 다른 장치 설정을 유지하는 것이 중요하다. 이미지는 획득 모듈의 터치 스크린 패널에 획득 버튼을 누름으로써 저장된다. </li><li> 6-15 분 후방에서 중간 단계의 이미지를 얻기어 사출. 참고 : 맥락막과 망막 혈관 모두가 뚜렷됩니다. 맥락막 혈관은 확산 형광을 나타납니다. 과 형광을 나타내는 맥락막 병변은 통상 배경 형광을 둘러싼 페이딩 대조적 등장. </li><li> 주사 후 17 ~ 25 분에서 후반 단계에서 이미지를 수집. 참고 :과 형광 페이드. 두 맥락막과 망막 혈관은 더 이상 볼 수 없습니다. 시신경의 머리는 검은 색이됩니다. 비침 맥락막 병변은 페이딩 배경으로 최대한의 대비를해야합니다. </li><li> 이미지의 인수를 마무리 한 후, 마우스의 눈에 분명 윤활유 눈 젤을 적용하고 복구를 위해 가열 패드에서 마우스를 둡니다. </li><li> 자신의 새장을 들고 지역에 마우스를 반환합니다. 추가 분석을 위해 TIFF 또는 JPEG 파일로 내보내기 이미지. </li></ol> 주 : 각 단계의 타이밍이 절대 아닙니다. 우리는 발견 EA의 타이밍CH상은 주입 ICG의 양에 따라 변경 될 수 있습니다. 더 ICG는 각 단계를 연장하는 경향이있다. 단계를 정의하는 가장 좋은 방법은 위에 나열된 각 단계의 주요 기능에 따라입니다.

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathology+of+age-related+macular+degeneration.">Molecular pathology of age-related macular degeneration.</a> Prog. Retin. Eye Res. 28, 1-18 (2009).</li><li>Grossniklaus, H. E., Kang, S. J., Berglin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+choroidal+and+retinal+neovascularization.">Animal models of choroidal and retinal neovascularization.</a> Prog. Retin. Eye Res. 29, 500-519 (2010).</li><li>Pennesi, M. E., Neuringer, M., Courtney, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+age+related+macular+degeneration.">Animal models of age related macular degeneration.</a> Mol. Aspects Med. 33, 487-509 (2012).</li><li>Elizabeth Rakoczy, P., Yu, M. J., Nusinowitz, S., Chang, B., Heckenlively, 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=Mouse+models+of+age-related+macular+degeneration.">Mouse models of age-related macular degeneration.</a> Exp. Eye Res. 82, 741-752 (2006).</li></ol>

YF는이 작품에 사용 된 AMD의 마우스 모델에 관한 두 개의 출원 특허의 발명가이다. SK, ZB, 그리고 ADJ는 공개 아무것도 없어.

이 연구에서, 우리는 HTRA1 형질 전환 생쥐의 이미지 맥락막 병변 ICGA의 사용을 보여 주었다. 우리의 마우스 모델에서 초, 중, ICGA의 후반 단계의 특징은 인간의 연구 1에서 잘 시간 코스와 일치합니다. 이는 AMD와 맥락막과 관련된 조건의 병태 생리 학적 기전 및 치료 전략에 대한 연구를위한 귀중한입니다 인간의 병리 및 동물 표현형 사이 좋은 비교를하는 것이 중요합니다. <p class="jove_con...

인도시 아닌 형광 안저 혈관 조영술 (ICGA)는 눈에 혈관과 관련된 이미지의 문제에 대한 진단 테스트입니다. 발광 스펙트럼은 770-880 nm 내지 835 nm의 1의 발광 피크와 범위 동안 ICG의 흡수 스펙트럼은 790-805 nm의 범위. 이것은 누구의 스펙트럼 가시 범위 내에 다른 인기있는 염료, 나트륨 형광 다릅니다. 적외선 스펙트럼은 쉽게 나트륨 형광 기반의 형광 혈관 조영술 (FA)로 시각화를 차단할 수있는 모두의 망막 색소 상피 세포 (RPE), serosanguineous 유체 및 지질 분비물을 통해 침투하는 ICG 수 있습니다. ICG는 맥락막 혈관과 맥락막 병변 1,2의 향상된 영상을 허용, 98 %의 단백질 바인딩 작은 혈관 외 유출의 결과로 혈관에 있습니다. ICGA 거의 RPE에 후방입니다 맥락막 혈관을 시각화 할 수있는 유일한 선택입니다. 1도 마우스의 눈에 영상 혈관의 ICGA와 FA의 비교를 보여줍니다. FA는 B 수이미지에 망막 혈관을 잘하지만 맥락막 혈관을 사용하는 전자. 대조적으로, ICGA 망막과 맥락막 혈관 양쪽 화상에 사용될 수있다. ICGA 함께 우리는이 연구에서 사용하는 적외선에 민감한 비디오 카메라와 고해상도 디지털 영상 시스템 또는 스캐닝 레이저 검안경 (SLO)으로 수행된다. 병원에서 ICGA는 결절 맥락막 혈관 병증 (PCV), 망막 Angiomatous 확산 (RAP), angioid 줄무늬, vitelliform 황반 영양 장애, 중심 장액 맥락 망막 병증, 맥락막 혈관종, 출혈, 망막 등의 맥락막 혈관을 포함하는 맥락 망막 질환의 수를 진단하는 권장 된 세동맥 macroaneurysms, 맥락막 종양, 후방의 특정 형태는 1,3 포도막염. FA 및 빛 간섭 단층 촬영 (10 월)와 ICGA의 조합은 삼출성 연령 관련 황반의 진단 및 관리에 임상의를위한 강력한 도구를 제공합니다변성 (AMD) 4-10. ICGA은 맥락막과 관련된 조건을 진단하는 데 특히 유용합니다. 사실, ICGA는 PCV, 삼출성 AMD 11 ~ 13의 변형을 진단하기위한 황금 표준으로 간주됩니다. PCV는 맥락막 혈관 11-13 터미널 결절의 확장이 혈관 분기의 네트워크를 특징으로한다. PCV 자주 누설 결절 구성 요소 11,14,15에서 출혈과 망막 색소 상피와 망막의 재발 serosanguineous 박리와 연결되어 있습니다. 우리는 최근 transgenically 마우스의 망막 색소 상피 (RPE) (16)에서, 사람의 HTRA1, 다기능 세린 프로테아제를 발현하여 제 PCV 동물 모델의 생성을보고 하였다. 우리는 증가 HTRA1가 PCV의 특징적인 기능, 예를 들어, 결절 병변을 유도 것으로 나타났다. 여기에서 우리는 우리의 HTRA1 마우스 모델을 사용하여 AMD의 연구에 꼬리 정맥 주사 시간 코스 ICGA의 사용을 보여 주었다. 우리의 데이터가 제안하는IV-ICGA는 IP (또는 피하 (SC)) 현재 맥락막 병변의 특성에 대한 필드 (17, 18)에 사용되는-ICGA에 우수합니다. 동물 연구에 문 동물 실험 기관 동물 관리 및 사용위원회 (IACUC)의 승인을 프로토콜에 따라 수행되었으며, 안과 및 비전 연구에서 동물의 사용에 대한 ARVO 정책에 따라 수행되었다.

<table border="0" cellpadding="0" cellspacing="0" width="667">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Spectralis Multi-Modality Imaging System</td>
			<td style="width:152px;">Heidelberg Engineering, Germany</td>
			<td style="width:132px;">SPECTRALIS HRA+OCT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tropicamide ophthalmic solution (1%)</td>
			<td style="width:152px;">Bausch &#38; Lomb</td>
			<td style="width:132px;">NDC 24208-585-64</td>
			<td>for dilation of pupils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenTeal Gel</td>
			<td style="width:152px;">Genteal</td>
			<td style="width:132px;">NDC 58768-791-15</td>
			<td>&#160;clear lubricant eye gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:152px;">Lloyd Laboratories</td>
			<td style="width:132px;">NADA 139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acepromazine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-160-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-G Needle</td>
			<td>Steriject</td>
			<td style="width:132px;">PRE-32013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-ml syringe</td>
			<td style="width:152px;">BD</td>
			<td style="width:132px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Indocyanine Green</td>
			<td style="width:152px;">Pfaltz &#38; Bauer</td>
			<td style="width:132px;">I01250</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting abnormalities in choroidal vasculature in a mouse model of age-related macular degeneration by time-course indocyanine green angiography

인도시 아닌 그린 혈관 조영술 (또는 ICGA)는 맥락막 및 연령 관련 황반 변성 (AMD)과 같은 각종 눈 질환의 망막 혈관의 이상을 진단하는 안과 의사에 의해 수행되는 기술이다. ICGA는 그것의 적외선 스펙트럼과 색소 층을 관통하는 자사의 능력에 눈의 뒤쪽 맥락막 혈관 이미지에 특히 유용합니다. ICGA 시간 과정은 초, 중, 후반 단계로 나눌 수 있습니다. 3 단계 눈 문제의 병리에 대한 유용한 정보를 제공합니다. 정맥 주사에​​ 의한 시간 코스 ICGA가 (IV) 주사가 널리 맥락막 문제의 진단과 관리를 위해 병원에서 사용되지만, ICGA은 복강 내 주사 (IP)에 의해 일반적으로 동물 연구에 사용됩니다. 여기에서 우리는 꼬리 정맥 주입 및 공 초점 주사 레이저 검안경에 의해 생쥐의 고해상도 ICGA 시간 코스의 이미지를 얻기 위해 기술을 보여 주었다. 우리는 맥락막 르 이미지에이 기술을 사용연령 관련 황반 변성의 마우스 모델에서 sions. 이 IP로 마우스 혈관에 ICG을 소개하는 것이 훨씬 쉽지만, 우리의 데이터는 IP-ICGA에 의해 재현 ICGA 시간 코스의 이미지를 얻기 어렵다는 것을 나타냅니다. 대조적으로, 꼬리 정맥 주사를 통해 ICGA 인간 연구에 비해 높은 품질의 ICGA 시간 코스의 이미지를 제공합니다. 또한, 우리는 ICGA가 착색 된 생쥐에서 수행보다 맥락막 혈관의 선명한 사진을 제공 흰둥이 생쥐에서 수행 것을 보여 주었다. 우리는 그 시간 코스 IV-ICGA 동물 모델을 기반으로 AMD의 연구의 표준 관행이 있어야 좋습니다.

우리는 CD1의 배경에 둘 중, HTRA1 형질 전환 마우스 및 제어 WT의 한배 새끼의 ICGA 시간의 과정을 수행 하였다. 흰둥이 CD1 배경은 인도시 아닌 형광 안저 혈관 조영술 (ICGA) 영상 (설명 참조)을 용이하게하기 위해 선택되었다. 의 확장이 같은 일부 동맥류가 HTRA1 마우스의 초기 단계에서 나타나기 시작했다 (그림 2, 빨간색 화살표는 용기의 끝 부분에 팽창을 나타내며 빨간색 원이 클러스터 ...

Watch this Scientific Journal Video about Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography at JoVE.com

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

꼬리 정맥 주입에 의해 수행 인도시 아닌 형광 안저 혈관 조영술 (ICGA 나)는 마우스 맥락막 이상을 특성화하기 위해 고품질의 ICGA 시간 코스의 이미지를 제공합니다.

시간 코스 인도시 아닌 형광 안저 혈관 조영술로 연령 관련 황반 변성의 마우스 모델에서 맥락막 혈관의 이상을 검출

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

detecting-abnormalities-choroidal-vasculature-mouse-model-age-related

Research

JoVE Journal

Medicine

13.6K 조회수.  University of Utah Health Sciences Center.  꼬리 정맥 주입에 의해 수행 인도시 아닌 형광 안저 혈관 조영술 (ICGA 나)는 마우스 맥락막 이상을 특성화하기 위해 고품질의 ICGA 시간 코스의 이미지를 제공합니다. 

비디오: Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

연구

의학

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

태아 알코올 노출의 Zebrafish 모델에서 Teratogenic 변경 사항 평가

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

마우스에있는 자기 공명 현미경 영상으로 실험자가 면역 심근염에있는 심장의 이상 유무 비 침습적 평가

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

레이저에 의한 맥락막 신생 혈관에 대한 마우스 모델

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

마우스에서 관절염 통증의 Monoiodoacetate 모델

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Anastasis에서 Vivo에서 CaspaseTracker 바이오 센서에 의해 감지

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

Suprachoroidal 세포 Autograft 건조 연령 관련 황 반 변성에 의해 재생 치료: 예비 Vivo에서 보고서

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Myography 와이어를 사용 하 여 마우스 Mesenteric 동맥의 isometric 수축 측정

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

광학 간섭 단층 촬영 혈관조영술로 측정된 황반 관류 밀도에 대한 모세관 및 기타 혈관 기여도 평가

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

괴사 장염의 신생아 BALB/c 마우스 모델

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...