윤리 문 : 모든 실험 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 동물 보호는 제도적 지침에 따라이었다. (- 8 주 오래 된 7; 25-30g) ICR 여성 마우스는 텔 아비브 대학에서 의학 학부 Sackler의 에어컨, 조명 제어 동물 시설에 수용되었다. 용어에서, 동물은 마취 과다 복용으로 안락사시켰다. 1. 섬유질의 공 초점 형광 현미경 (FCFM) 교정 <ol><li> 장치를 켜십시오. </li><li> 마이크로 탐침 (mini0이 / 30)을 연결합니다. </li><li> 제조업체의 지시에 따라 교정 장치. </li></ol> 2. 마우스 이미징을위한 준비 <ol><li> 모두 Ketaset (100 ㎎ / ㎏)과 XYL-M2 (6 ㎎ / ㎏)의 피하 주사에 의해 마취. 발가락 핀치에 응답하지 않는 문제로 적절한 마취를 확인합니다. </li><li> 순서에 사타구니 아래 피부를 절개<html> 대퇴 동맥 혈관을 알 수있다. 절개 다음 식염수 촉촉한 절개 사이트를 유지합니다. </li><li> 약 30 초 동안 따뜻한 물 (너무 뜨겁지 않은 터치)로 가득 찬 가방 (또는 장갑)를 사용하여 꼬리를 가열. 식염수 또는 화학 요법 제 중 하나의 꼬리 정맥에 주사 바늘 (30 G, 1/2 인치)를 삽입하고 여기에 1 ML의 주사기를 부착하여 FITC 덱스 트란 (조영제)의 관리를위한 정맥 주사 (IV) 션트를 준비하고 . 정맥은 식염수를 주입하여 열려 있는지 확인하십시오. 참고 : FITC 덱스 트란의 IV 관리 (고 분자량 100 ㎕의 10 ㎎을 / ㎖, 2000 kDa의)이 FCFM에 의한 대퇴 미세 혈관을 시각화 용이. 독소루비신 (100 μL; 8 ㎎ / kg, 아드리아 마이신) 또는 염수 나중에 예열 꼬리 정맥으로 IV 투여 될 것이다. </li><li> 폴리스티렌 무대에 드러 마우스를 놓습니다. 패드에 마우스를 안전하고 수술 덕트 테이프를 사용하여 위치를 유지한다. </li></ol>동안과 독소루비신 또는 식염수를 투여 한 후 FCFM에 의한 대퇴 혈관의 제목 &quot;&gt; 3. 영상 참고 : (1) 마이크로 탐침 (mini0이 / 30) :이 연구에 사용 된 다발, 공 초점 현미경은 두 개의 유닛으로 구성되어있다. (2) 레이저 스캐닝 유닛 (LSU-488; 488 nm 파장). <ol><li> LSU 488 nm 파장의 레이저를 이용하여 분석 모든 시간 바퀴를 수행합니다. 참고 : 본체 검출기는 여과 감지 (500-650 nm의) 방출 형광을. 획득 된 이미지는 이후 재구성 / 초 12 프레임의 속도로 표시됩니다. </li><li> 조심스럽게 바늘의 주사기를 분리하고 FITC 덱스 트란을 포함하는 새로운 주사기를 연결합니다. FITC 덱스 트란의 (IV) 100 μl를 관리합니다. </li><li> 대응하는 화상을 얻기 위해서, Z 축 조정 다음에, 뷰 필드에 적합한 마이크로 프로브 (mini0 / 30)를 시프트하고 흥분시키는. 명확하고 초점을 맞춘 신호가 VISI 때까지 페이드 초기 신호를 기다립니다BLE. </li><li> 짧은 안정화 기간 (~ 30 초)에 대한 기준선 혈액의 흐름을 기록합니다. 그런 다음 독소루비신 또는 식염수를 포함, 바늘에 다른 주사기를 연결합니다. IV 100 μL 독소루비신 또는 식염수를 관리 할 수​​ 있습니다. </li><li> 20 분 동안 연속적으로 주입 FITC 덱스 트란의 흐름을 모니터링한다. FCFM 관련된 소프트웨어를, 혈관을 측정하기 위해 상부에 눈금자 직경 버튼을 사용하여 분류하고 그들 작은 (&lt;15㎛ 인) 또는 큰 (&gt; ~ 15㎛). </li><li> 마취 과다 복용으로 동물을 안락사. </li></ol>

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화학 요법 - 유도 된 혈관 독성을 평가하기 때문에 실시간으로 자극에 반응하여 혈관의 역학을 시각화하는 어려움에 도전. 수많은 임상 연구는 여러 가지 화학 요법 직접 혈관 손상을 일으킬 것으로 연루, 아직이 독성의 메커니즘과 특성은 밝혀지지 남아있다. 우리는 여기에 8 ~ 10 설명 된 바와 같이 다발, 공 초점 형광 현미경으로 구성된 마우스에서 화학 요법의 잠재적 인 혈관 독성을 평...

임상 증거는 화학 요법의 여러 클래스가 레이노 현상, 고혈압, myocardialinfarction, 뇌 혈관 공격, 간성 veno-occlusivedisease 1, 2에 의해 나타난 혈관 병변의 다양성을 이끌어내는 것을 나타냅니다. 그들은 원래 이러한 목적 3-5으로 개발되지만 적게 부과함으로써 종양 세포를 제거하도록 설계되지는 않았지만 &quot; &#39;사고&#39;항 혈관 형성 약제&quot;는 &quot;담보 가능한 혈관 신생 억제제로서 작용 종래의 화학 요법 제에 대해 설명 신종 용어이며 가능한 3과 정상 세포의 손상 &quot;. 혈청 바이오 마커를 사용하여 임상 연구에서 관찰 된 여러 화학 요법은 vasculo - 독성 물질로 암시되고있다. 이 중 에이전트 (예 : 시클로 포스 파 미드 등), 백금 (시스플라틴 등) 화합물 및 안트라 사이클린 1,2,5-7 알킬화된다. 급성 심장 혈관 합병증은 resul로 발생할 수 있습니다화학 요법에 의해 유도 된 혈관 독성 t. 그들은 늦게 혈관 사망률의 위험이 증가 동맥 경화 및 계정 등의 만성 질환으로 발전 할 수 있습니다. 그러나 임상 적 증거를 장착에도 불구하고, 혈관 독성의 메카니즘을 강조 지정된 연구가 소수이므로, 그들은 입힐 정확한 병인의 추가 해명이 보증된다. 화학 요법에 의한 혈관 독성의 메커니즘을 밝히는데 중요한 과제는 생체 내에서 혈관 기능을 조사의 복잡성에서 유래. 우리는 본원에서 혈류 및 혈관 &#39;특성을 포착 할 수 있도록 마우스에서 생체 분자 이미징에 대한 고해상도의 플랫폼을 설명한다. 이 플랫폼은 직접 유도 혈관 치료 효과의 검출을 용이하게한다 : 실시간뿐만 동일인 내의 일정 기간 동안 이들을 다음과 같이.

<table border="0" cellpadding="0" cellspacing="0" width="1395">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:579px;">Name of Material/ Equipment</td>
			<td style="width:423px;">Company</td>
			<td style="width:213px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">general anesthesia (100mg/kg ketaset and 6mg/kg XYL-M2).&#160;</td>
			<td>Fort Dodge Animal Health, IA, USA and Biove Laboratories, France</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">a depilatory cream (Veet)</td>
			<td>&#160;ReckittBenckiser, Bristol, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A 30-gauge, 1/2-inch needle attached to 1 ml syringe&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC dextran at a volume of 100 &#181;l (10 mg/ml; FD2000S, MW 2000000 Dalton)&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxorubicin (8 mg/kg, Adriamycin)</td>
			<td>Teva, Israel</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paclitaxel (1.2 mg/kg, Medexel)&#160;</td>
			<td>Taro, Israel</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

chemotherapy-induced vascular toxicity - real-time in vivo imaging of vessel impairment

화학 요법은 특정 부류의 병적 혈관의 증가 된 위험에 걸리기 쉬운 환자 수 장기적인 상태로 진행​​할 수있다 급성 혈관 변화를 가할 수있다. 그러나, 장착 임상 증거에도 불구하고, 혈관 독성 때문에 혈관 / 심혈관 질환의 이종 그룹의 원인의 명확한 연구의 소수는 밝혀지지 남아있다. 또한, 혈관 독성의 기초가있다 메커니즘은 완전히 심근 손상을 유도하는 관련된 화학 요법 - 유도 심독성의 원리와 다를 수있다. 우리는 항암 치료의 잠재적 급성 혈관 독성을 평가하는 생체 분자 이미징 플랫폼에서 실시간을 확립했다. 우리는 적절한 밀폐 된 기관 및 참조 BLO 내 혈관을 시각화 생체, 마우스의 고해상도 분자 영상의 플랫폼을 설정 한각 개별 반면 같은 개인 내 외경 선박은 자체 제어 역할을합니다. 혈관벽 최종 장기 손상을 조기 이벤트가 될 수있다 혈관 독성을 나타내는 고유기구, 독소루비신 투여 후 손상되었다. 여기서, 다발, 공 초점 형광 현미경 (FCFM) 기반 이미징의 방법은 동물 피험자에서 세포 및 세포 이하 수준에서의 생리 현상을 이해하기 위해 혁신적인 모드를 제공하는 설명한다.

실시간 생체 내 이미징 연속 여기에 사용되는 촬상 장치는, 혈관계의 시각화 및 화학 요법과 같은 다양한 자극에 대한 응답을 가능하게 장착 된 프로브 고화질, 다발, 공 초점 현미경이다. 깊숙히 혈관 또는 기관의 영상을 용이하게 할 수 있지만,이 프로브 작은 절개를 필요로하기 때문에이 방법은 침습적이다. 프로브 번들은 수십 섬유, 현미경 광학 및 독점 정밀 커넥터?...

Watch this Scientific Journal Video about Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment at JoVE.com

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

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

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

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

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Medicine

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

7.3K 조회수.  Tel Aviv University. We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

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

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Gynecologic 종양학의 센티널 림프절의 Intraoperative 감지 Multispectral 실시간 형광 이미징

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

연구

의학

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Intravital 이미징을 사용하여 실시간으로 종양의 Nanoparticle의 이해 평가

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

<em&gt; 생체 내</em혈관 투과율을 테스트하려면&gt; 분석

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

길이, 비 침습적을위한 안구의 앞쪽 상공 회의소에 이식<em&gt; 생체에</em실시간으로 단일 셀 해상도&gt; 영상

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

신생아 마우스 폐에서 폐 유체 볼륨의 실시간 X 선 영상

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

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

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

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

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

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

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

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

철 염화물에 의한 경동맥 및 장간막 용기에 혈전증 마우스 모델

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

종양 미세 환경에서 실시간 다세포 역학의 확장 시간 경과 생체 내에 영상

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

인간 태반 생리학의 통합 연구에 대한 4 혈관 샘플링 접근법<em&gt; In Vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

쥐의 급성 심근 경색의 실시간 압력-볼륨 분석

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...