eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Intravital Imaging
NOTE: The time interval between the tumor cell injection and the first intravital imaging session is dependent on the type of tumor cell line used. For the experiments shown in this protocol, 1x 105&#160;GL261 cells were injected and imaged 10 days later.
<ol>
	<li>Imaging preparation
	<ol>
		<li>Sedate the mouse using isoflurane inhalation anesthesia through a face mask (1.5%&#8211;2% isoflurane/O2&#160;mixture).</li>
		<li>Inject the mouse subcutaneously with 100 &#181;L of saline buffer to prevent dehydration. 
		NOTE: For long-term imaging, the mouse can be hydrated through a subcutaneous infusion pump.</li>
		<li>Place the mouse face-up in an imaging box. Use a metal plate with a 1 mm-diameter hole and small magnets embedded around the aperture to provide fixation of the CIW to the imaging box. Introduce isoflurane through a facemask and ventilate by an outlet on the other side of the box (0.8%&#8211;1.5% isoflurane/O2&#160;mixture). Optionally, use tape to fix the body of the mouse. 
		NOTE: Fluorescently labeled dextran for blood vessel visualization or other dyes may be injected intravenously at this point.</li>
		<li>Optionally, use a pulse oximeter and a heating probe to monitor the mous&#39;s vitals. 
		NOTE: In this experiment, it was not necessary to use a pulse oximeter and a heating probe since the mouse was stable throughout the imaging time period (2&#8211;3 h). However, for longer imaging times, more thorough monitoring may be needed.</li>
		<li>Set the 25x (e.g., HCX IRAPO NA0.95 WD 2.5 mm) water objective to the lowest&#160;z-position and add a large drop of water. 
		NOTE: The use of a water immersion microdispenser is highly advised for long term experiments since it allows scientists to add water during the experiment. Alternatively, a dry objective can be used.</li>
		<li>Transfer the imaging box onto the microscope equipped with a dark climate chamber kept at 37 &#176;C. Bring the objective to the CIW coverslip until the water drop touches it.</li>
		<li>Using the epifluorescence mode, observe the tumor through the eyepiece and bring the cells into focus.</li>
	</ol>
	</li>
</ol>
2. Time-lapse image acquisition
<ol>
	<li>Select several positions of interest to image and record their coordinates in the software. Ensure that the selected positions are representative positions from different sites of the tumor (each tumor can be different, but to ensure consistency, select the same amount of positions that are central to the tumor core and to the edges across all mice). 
	NOTE: A tile scan of a part of the tumor or of the whole visible tumor may be performed; however, if the tumor is large, this method will increase the time-lapse between images. In addition, if the tumor cells move fast, it can be challenging to track the same cells over time.</li>
	<li>Switch to multiphoton mode and tune the laser to the correct wavelength. Note that to avoid photodamage, higher wavelengths are desired. 
	NOTE: For Dendra2 imaging, a 960 nm wavelength was used. With the objective used in these experiments, a zoom of 1.3 was sufficient to get a good resolution of the tumor nuclei and scan a representative area of the tumor.</li>
	<li>Go to the live mode and define a&#160;z-stack for each position in order to acquire the maximal volume of tumor cells without compromising the tumor cell resolution. Define the step size between the images as 3 &#181;m.</li>
	<li>Use the bidirectional mode to increase the scanning speed. The image resolution should be at least 512 x 512 pixels.</li>
	<li>Acquire images of the tumor volume at different positions every 20 min for 2 h. Add water to the objective before each image acquisition. 
	NOTE: Time-lapse images can be automatically acquired by setting the right time-lapse in the software. However, the mouse can move, and the position or&#160;z-stack can shift over time, causing data loss. Therefore, it is recommended to perform the time-lapse manually and to check and adjust for&#160;xyz&#160;shifts in between acquisitions.</li>
	<li>At this step, optionally, photo-switch the Dendra2 fluorescent marker. 
	NOTE: As opposed to time-lapse imaging (which allows studying individual tumor cells properties and how they change over time), this will allow studying the area of tumor cell infiltration in the brain over several days.</li>
</ol>

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			<td>Opthalmic ointment&#160;&#160;</td>
			<td>Kela Veterinaria</td>
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cranial window-based intravital imaging in a mouse model: an imaging technique to study the behavior of pre-injected cancer cells in the brain of a murine model

Source: Alieva, M. and Rios, A. C. <a href="https://www.jove.com/v/59278/longitudinal-intravital-imaging-brain-tumor-cell-behavior-response-to">Longitudinal intravital imaging of brain tumor cell behavior in response to an invasive surgical biopsy.</a>&#160;J. Vis. Exp.&#160;(2019)
In this video, we perform intravital imaging of pre-injected cancer cells in a mouse model via an implanted cranial window. This&#160;high-resolution time-lapse imaging technique enables the study of dynamic behavior of cancer cells in the live mouse.

Cranial Window-based Intravital Imaging in a Mouse Model: An Imaging Technique to Study the Behavior of Pre-injected Cancer Cells in the Brain of a Murine Model

Source: Alieva, M. and Rios, A. C. Longitudinal intravital imaging of brain tumor cell behavior in response to an invasive surgical biopsy.&#160;J. Vis. ...

Intravital imaging through optical imaging windows facilitates high-resolution tracking of individual cells to study their behavior in the native microenvironment in a live animal.
To begin, take an anesthetized mouse with its brain pre-injected with cancer cells and having an implanted cranial window fit inside a metal ring. The cancer cells express nuclear fluorescent proteins.&#160;
Position the mouse, face up, in an imaging box. The magnetic holder in the imaging box helps secure the implanted cranial window.
Now, transfer the imaging box inside the environmentally controlled imaging chamber of an inverted microscope to analyze the behavior of cancer cells in the brain.
Under multiphoton mode, set the laser to a suitable wavelength. Focus on the tumor region to detect the fluorescence from cancer cells.&#160;
Define a z-stack for each position to acquire multiple intravital images of cancer cells in different optical planes.
This step enables the capture of a significant proportion of cancer cells in the tumor microenvironment without affecting cell resolution.
Subsequently, acquire images from different positions of the tumor for an extended duration.
Tracking the path of cancer cells in different optical planes helps determine individual tumor cell migration using suitable software.

cranial-window-based-intravital-imaging-mouse-model-an-imaging

Research

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

In vivo studies

1.2K ビュー. 出典:Alieva, M. and Rios, A. C. 侵襲的外科的生検に応答した脳腫瘍細胞の挙動の縦断的生体内イメージング。J. Vis. Exp.(2019年) このビデオでは、マウスモデルに事前に注射されたがん細胞の生体内イメージングを、埋め込み頭蓋窓を介して行っています。この高解像度タイムラプスイメージング技術により、生きたマウスにおけるがん細胞の動的挙動の研究が可能になります。 

ビデオ: マウスモデルにおける頭蓋窓ベースの生体内イメージング:マウスモデルの脳内における事前注射癌細胞の挙動を研究するためのイメージング技術 - 概念

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

インビタルイメージングと固定組織解析を用いた癌細胞播種に伴う転移戸管媒介血管透過性の腫瘍微小環境の評価

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

JoVE Journal

がん研究

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

落射蛍光による組織マトリックスのコンポーネントと二光子顕微鏡の長期生体内免疫蛍光イメージング

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

生物工学

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

膵臓内イメージングウィンドウを用いたマウスモデルにおける膵臓の細胞レベル可視化におけるビ ボ における縦方向の安定化

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Cranial Window-based Intravital Imaging in a Mouse Model: An Imaging Technique to Study the Behavior of Pre-injected Cancer Cells in the Brain of a Murine Model

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