We thank Drs. Xiaofang Wang and Katharina Hopp for their invaluable help with the animal models. This work has been supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK090728, DK058816).

살아있는 동물과 어떤 절차를 시작하기 전에 실험 프로토콜은 기관 동물 관리 및 사용위원회 (IACUC)의 승인을 받아야한다. 1. 스캐너 구성 <ol><li> 시작하기 전에, 히터가 OFF 위치에 있는지 확인하십시오. </li><li> 미니 이미징 그라데이션과 38mm의 RF 코​​일과 미니 영상 홀더를 선택합니다. </li><li> 홀더의 중심 보어 가변 온도 어셈블리를 설치한다. </li></ol> 2. 동물 준비 <ol><li> 자기 공명 영상 실험을 위해, 증발 이소 플루 란을 사용하여 최적의 마취를 얻을 수 있습니다. 마취의 유도, 흡수 조직 늘어서 유도 챔버에서 동물을 배치합니다. 2.0-2.5 L / 분, 산소의 3 %에 이소 플루 란에 이소 플루 란 기화기의 유량계를 조정합니다. </li><li> 이 단계에서 금속 태그 또는 다른 금속 물체를 제거합니다. 마취 동안 건조를 방지하기 위해 동물의 눈에 수의사 연고를 적용합니다. </li><li> 동물 일단마취의 수술 비행기에 도달했습니다 (즉, 발가락 핀치에 철수 반사 손실)은, 코 콘에 삽입 코와 홀더에 동물을 배치합니다. 2.0-2.5 ml / 분에 프로브 마취 공기 흐름과 산소의 1.5-2.0 %로 이소 플루 란 농도를 설정합니다. 마취 절차를 수행하는 동안 코 콘을 통해 전달됩니다. 주기적 ~ 40 BPM의 호흡 속도를 유지하기 위해 동물의 연령 및 체중에 따라 이소 플루 란 농도를 조정한다. </li><li> 대신에, 동물을 고정하고 MRI 실험 동안 움직임을 방지하기 위해 동물 홀더를 사용. 바디 영역에 따라 동물 홀더 유형이 스캔 할 다양하다. 주 : 실험실 플라스틱 (폴리 프로필렌, 테플론, 폴리스티렌, 폴리 카보네이트)로부터 받아서 홀더 특정 실험을 수용하고 (160g 래트 신생아로부터 마우스) 동물의 크기에 맞게 만들어 질 수있다. </li><li> 동물의 신체 온도를 모니터링하기 위해 동물의 직장 온도계를 놓습니다. 특급시eriment 따뜻한 공기의 스트림을 사용하여, 35-37 ℃에서 동물 유지. 공기 온도 (30-38 ° C의)과 동물의 체온 피드백을 기반으로 흐름 (1,200-2,000 리터 / h)를 조정합니다. </li><li> 호흡 속도를 모니터링하는 동물의 복부에 풍선 호흡 압력 센서를 부착합니다. </li><li> RF 코일의 중심에서, 동물을 고정하고 신중 MRI 스캐너로 동물 RF 코​​일을 배치했다. </li></ol> 3. 자기 공명 영상 실험 <ol><li> 조정이 실험은 RF 전력 사용을 최소화하고, 신호 대 잡음비를 최대화하기 시작하기 전에 RF 코​​일 일치. 일치 / 튜닝을 시작하려면 : <ol><li> 도구 아이콘을 클릭하여 분광계 제어 도구를 엽니 다. </li><li> 분광계 제어 도구에서 수집 → 워블을 클릭합니다. ACQ / 레코 창 워블 곡선을 표시 열립니다. </li><li> 선택적 반사 RF 전력까지 작은 단계로 조정 및 정합 커패시터 (튜닝 봉을 사용하고 매칭)을 조정할최소화된다. 목표는 횡축 제로에 위치 수직축에서 최소 곡선을 볼 수있다. </li><li> 코일의 교정이 성공적으로 달성되면, ACQ / 레코 창에서 정지 버튼을 누르십시오. </li></ol></li><li> , 축 관상 및 시상 이미지를 만드는 세 직교면에서 스카우트 이미지를 획득. 스카우트 이미지 (12)를 취득하는 등 내부 게이트 빠른 낮은 각도 샷 (IG-FLASH)와 같은 빠른 이미지 시퀀스를 사용합니다. 실제 영상에 대한 적절한 형상을 설정하기 위해 스카우트 이미지를 사용합니다. </li><li> 특정 연구 목적에 따라 적절한 이미지 시퀀스 및 매개 변수를 선택하고 신호등과 스캔을 시작합니다. 이것은 모든 자동, RF 채널을 교정 물에 공명 자석, 설정된 반송파 주파수를 심은 및 수신기 이득을 조정한다. <ol><li> 해부학 적 연구 및 T2 강조 영상의 경우, 2D 멀티 슬라이스 또는 3D 모드로 취득. 필드의 유지, 주어진 공간 해상도 실험 시간을 단축의 클로 뷰 (FOV) 가능하지만 랩 어라운드 유물 (2.56-3.2 CM)을 방지하기에 충분히 큰만큼 작은. </li></ol></li><li> 반복 시간 (TR) 및 / 또는 조각의 수의 적절한 선택에 의해 동물의 호흡주기보다 약간 짧은 선택한 시퀀스의주기를 유지합니다. 이 데이터는 동물의 조용한 기간 동안 수집되는 것을 보장한다. <ol><li> 예를 들어, 복부 이미지, ~ 30 BPM에서 동물의 호흡을 유지; 그 숨 당 약 2,000 밀리 초입니다. TR로, 휴식 향상 (RARE) 순서로 터보 빠른 취득을 사용하여 11-19 관상 조각을 취득 / TE 9분의 1,500 밀리 초 희귀 인자 8 (매트릭스 256 X 256, FOV 2.56 X 2.56 cm, 슬라이스 두께 0.75 ㎜) . 참고 : 1,500 밀리 초에 TR을 조정하고, ~ 30 BPM을 (숨 2,000 밀리 초) 동물의 호흡 속도를 유지함으로써, 우리는 데이터가 동물의 조용한 기간 동안 수집되어 있는지 확인합니다. </li></ol></li><li> 모든 화상 취득이 완료되면, 주사 동물을 배치가열 패드에 외래 때까지 모니터링 할 수 있습니다. 복구 후 케이지에 동물을 반환하고, 동물 시설로 돌아 가기 전에 1 시간 동안 적어도 모니터링. </li></ol>

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The authors have nothing to disclose.

이 원고는 생체 표현형 특성 또는 PKD에 대한 설치류 모델에서 약물 모니터링을위한 도구로 UHF 자기 공명 영상 사용의 가능성을 보여줍니다. 우리는 넓은 구멍 마이크로 미니 이미징 액세서리를 갖춘 이동이다 III 고해상도 NMR 분광계와 16.4 T에서 수행 실험을 설명합니다. 분광계는 Paravision 5.1 이미징 소프트웨어에 의해 제어 TopSpin2.0PV 취득 및 처리 소프트웨어에 의해 ?...

다낭성 신장 질환 (PKD)는 신장 낭의 발달을 특징으로 단일 유전자 질환의 군을 포함한다. 그 중 상 염색체 우성 다낭성 신장 질환 (ADPKD)과 염색체 - 열성 다낭성 신장 질환의 가장 일반적인 유형 1, 2를 나타냅니다 (ARPKD)이다. ADPKD, 유전성 신장 낭종 성 질환의 가장 흔한 형태는 PKD1 또는 PKD2 유전자의 돌연변이에 의해 유래. 그것은 후기 발병, 여러 양측 신장 낭종, 변수 여분 신장 낭종을 동반뿐만 아니라 심장 혈관과 근육 골격의 이상을 특징으로한다. ARPKD, 가장 일반적으로 영향을 미치는 신생아 및 어린 아이들, PKHD1 돌연변이에 의해 발생 및 확대 에코 신장과 선천성 간 섬유증 3을 특징으로한다. 중요한 것은, ADPKD 모두 상당한 P 결과 유전자 (제닉)과 변이 (대립 유전자) 수준에서 이질성이 특징입니다henotypic 변화. PKD1 유전자의 돌연변이는 심한 임상 양상 (다수의 낭종, 조기 진단, 고혈압, 및 혈뇨),뿐만 아니라 (PKD2 돌연변이를 가진 환자는 20 년 전) 신장 질환 단계를 종료하는 빠른 진행과 관련된 4. 심한 다낭성 간 질환 (PLD)과 혈관 이상은 PKD1과 PKD2 5 모두에 돌연변이와 연관 될 수 있습니다. ADPKD의 신장 합병증의 대부분은 연관된 염증 및 섬유증과 함께 낭종 팽창의 결과로 주로 발생한다. 낭종 개발은 자궁에서 시작하여 환자의 일생을 통해 계속됩니다. 신장은 보통 20 개 이상의 배 정상 신장 볼륨에 도달 할 수에도 불구하고 자신의 콩팥 모양의 모양을 유지한다. 신장 낭종의 환자 현재 양측 분포의 대부분은, 그러나 어떤 특별한 경우에, 낭종은 일방적 또는 비대칭 패턴으로 개발할 수 있습니다. 주요 challen신장 내과는 ADPKD 환자를 다음 또는 치료를 구현하기위한 GE는 질병의 자연 경과입니다. 그 과정의 대부분 동안, 신장 기능은 정상 유지 및 신장 기능이 감소하기 시작하는 시간, 신장의 대부분은 낭종에 의해 대체되었습니다. 치료법은 나중 단계에서 구현 될 때, 이는 환자가 이미 만성 신장 질환의 귀환 포인트에 도달 할 수 있기 때문에 성공하기 어렵다. 치료는 초기 단계에서 시작되는 경우 반대로, 그것은 전적으로 사구체 여과율에 기초한 응답을 식별하는 것이 곤란하다. 그 결과, 질병 진행의 마커로서 신장 용적의 개념은 주목을 받았다. ADPKD 환자에서 신장 낭종 볼륨의 증가는 직접 한 총 신장 볼륨 (TKV)의 잠재력을 강조, 신장 기능 저하와 관련이 있음을 다낭성 신장 질환 (선명한) 연구의 방사선 영상 연구 컨소시엄을 보여 주었다질병 진행의 6,7에 대한 urrogate 마커. 따라서, TKV 현재 ADPKD 2,8,9에 대한 여러 임상 시험에서 기본 또는 보조 엔드 포인트로 사용됩니다. 여러 쥐 모델을 포함 자발적인 돌연변이와 유전자 조작은 PKD 10, 11의 발병 기전을 밝혀왔다. Pkd1 또는 Pkd2 모델 (Pkd1 또는 Pkd2 중 하나에 돌연변이), 그들이 완벽하게 모방 인간의 질병에서 가장 인기있는 사람이되었다. 또한 Pkd1 또는 Pkd2 유전자 이외의 유전자 돌연변이 쥐 모델 질환과 관련된 신호 전달 경로 해명 실험 플랫폼으로 사용되어왔다. 또한, 이들 모델의 몇몇 잠재적 인 치료법을 테스트하는 데 사용되어왔다. 그러나, PKD 많은 설치류 연구에 제한 요소를 순차적으로 신장 해부학 적 및 기능적 변화를 분석하는 효율적인 비 침습적 방법은 종종 부족하다. 자기 Resonance 이미징 (MRI)이 우수한 부드러운 티슈 콘트라스트 및 해부학 상세를 제공하고 있으나 TKV 측정을 허용 ADPKD 환자를 따르는 전류 금 표준 영상 법이다. MRI가 아니라 생체 내에서 더 큰 동물과 인간, 촬상 작은 설치류에서 해부학 촬상 수립 되더라도 높은 해상도의 이미지를 획득 할 수있는 능력이 그 유용성을 제한 할 수있는 추가의 기술적 도전을 수반한다. 초고 필드 (UHF) MRI (7-16.4 T)과 강한 구배 현상의 도입으로, 이것은 유사한 잡음비 및 진단 품질 MRI 화상의 공간 해상도 신호 - 더 달성하는 것이 가능하다 인간에서 얻어진. 따라서, PKD에 대한 작은 설치류 모델의 생체 내 이미징을위한 UHF MRI의 사용은 연구를위한 강력한 도구가되고있다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AVANCEIII-700 (16.4 T)</td>
			<td>Bruker</td>
			<td>BH067206</td>
			<td>Wide-bore two channel multinuclear spectrometer equipped with mini and micro-imaging accessories for in vivo small rodent imaging</td>
		</tr>
		<tr>
			<td>TopSpin2.0PV&#160;</td>
			<td>Bruker</td>
			<td>H9088TA2</td>
			<td>Spectrometer processing software&#160;</td>
		</tr>
		<tr>
			<td>Paravision 5.1&#160;</td>
			<td>Bruker</td>
			<td>T10314L5</td>
			<td>Imaging sofware</td>
		</tr>
		<tr>
			<td>VTU BVT 3000 digital</td>
			<td>Bruker</td>
			<td>W1101095</td>
			<td>Temperature controller</td>
		</tr>
	</tbody>
</table>

use of ultra-high field mri in small rodent models of polycystic kidney disease for in vivo phenotyping and drug monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

이 논문에서 우리는 PKD 및 기타 신장 질환에 대한 설치류 모델에서 생체 표현형 특성 또는 약물 모니터링을위한 도구로 UHF 자기 공명 영상의 유용성을 표시하는 것을 목표로하고 있습니다. 모든 실험은 IACUC 승인 실험 프로토콜의 일부였다. PKD는 UHF MRI를 사용하기위한 작은 설치류 모델의 생체 표현형에서 : 모든 영상 검사는 브루 커 AVANCEIII-7...

Watch this Scientific Journal Video about Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring at JoVE.com

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

대한 다낭성 신장 질환의 작은 설치류 모델에서 매우 높은 필드 MRI의 사용<em&gt; 생체</em&gt; 표현형 의약품 모니터링

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

use-ultra-high-field-mri-small-rodent-models-polycystic-kidney

Research

JoVE Journal

Medicine

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

11.4K 조회수.  Mayo Clinic. The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

비디오: Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

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 ...

연구

의학

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Polycystic 신장 질환과 함께 인간과 마우스의 염색체 이상을 공부하는 스펙트럼 Karyotyping

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

파킨슨 병 분야에서 Mitochondrial Phenotypes을 모니터링하기위한 기본 인간 섬유 아세포의 사용

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

작은 동물 룩 로커 반전 복구 (SALLI) 쥐의 MRI를 사용하여 심장 기능 및 심근 형태학의 평가

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

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. ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

다낭성 신장 질환의 가능한 제브라 피쉬 모델 :의 넉다운<em&gt; wnt5a</em&gt; Zebrafish의 신장의 낭종 원인

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

예 생체 내 창자 주머 니나는 위장 질환 모델에서 점막 투과성을 평가하는

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

양적 3D<em&gt; 인 실리코</em알츠하이머 병의 쥐 모델에서 뇌 아밀로이드 - 베타 탐식&gt; 모델링 (q3DISM)

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Vivo에서 골절 뼈 쥐 마우스 대 퇴 골에 대 한 MRI-호환 Osteosynthesis 장치를 사용 하 여 치료 기간 동안 굳은 살 개발의 평가

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...