Name: Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment (Video) | JoVE Journal
Uploaded: 2015-01-07T13:00:00.000+00:00
Duration: 4 min 48 s
Description: Learn fibered confocal fluorescent microscopy for real-time vascular imaging in live animal models. This method enables evaluation of chemotherapy-induced vascular toxicity using fluorescent dextrin visualization.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

playerTrialDesktop

Ethics statement: All experiments were approved by the Institutional Animal Care and Use Committee. Animal care was according to institutional guidelines. ICR female mice (7 - 8 weeks old;&#160;25 - 30 g) were housed in air conditioned, light controlled animal facilities of the Sackler Faculty of Medicine in Tel-Aviv University. At term, animals were euthanized with anesthesia overdose.
1. Fibred Confocal Fluorescence Microscopy (FCFM) Calibration
<ol>
	<li>Turn the device ON.</li>
	<li>Connect the microprobe (mini0/30).</li>
	<li>Calibrate the device according to manufacturer&#8217;s instructions.</li>
</ol>
2. Mice Preparation for Imaging&#160;
<ol>
	<li>Anesthetize&#160;by a subcutaneous injection of both Ketaset (100 mg/kg) and&#160;XYL-M2 (6 mg/kg). Confirm proper anesthetization by unresponsiveness to toe-pinch.</li>
	<li>Incise the skin&#160;below&#160;the&#160;groin&#160;in order to&#160;reveal&#160;the femoral arterial vessels. Keep the incision site moist with saline following incision.</li>
	<li>Heat the tail by using a bag (or a glove) filled with warm water (not too hot to touch) for approximately 30 sec. Prepare an intravenous (IV) shunt for administration of FITC dextran (a contrast agent) and of either saline or chemotherapeutic agent, by inserting a needle (30 G, 1/2 inch) into&#160;the&#160;tail vein and attaching a 1 ml syringe to it. Ensure the vein is open by injecting saline. 
	NOTE: An IV administration of FITC dextran (high molecular weight; 100&#160;&#181;l; 10 mg/ml; 2,000 kDa)&#160;facilitates visualizing the femoral microvasculature by FCFM.&#160;Doxorubicin (100 &#181;l; 8 mg/kg, Adriamycin) or saline&#160;will also be later&#160;administered IV into the pre-heated tail vein.</li>
	<li>Position the mouse supinely on a polystyrene stage. Secure the mouse to the pad and maintain position using surgical duct tape.</li>
</ol>
3. Imaging of Femoral Blood Vessels by FCFM During and After Administration of Doxorubicin&#160;or Saline
NOTE: The fibered confocal microscope used in this study is composed of two units: (1) microprobe (mini0/30). (2) Laser scanning unit (LSU-488; 488 nm wavelength).
<ol>
	<li>Perform all time-laps analyses using the LSU 488 nm wavelength laser.&#160; 
	NOTE: The main unit detector detects the filtered (500 - 650 nm) emitted fluorescence. The acquired images&#160;are&#160;reconstructed afterwards and displayed at&#160;a&#160;rate of 12 frames/sec.</li>
	<li>Disconnect carefully the syringe from the needle and attach a new syringe containing FITC dextran. Administer (IV) 100 &#181;l of FITC dextran.</li>
	<li>Shift&#160;the microprobe (mini0/30) to a suitable field of view and fixate it, following adjustment to the z-axis, in order to obtain the corresponding image. Wait for the initial signal to fade until a clear and focused signal is visible.</li>
	<li>Record a baseline blood flow for a short stabilization period (~30 sec). Then connect to the needle another syringe, containing either doxorubicin or saline. IV administer 100 &#181;l doxorubicin or saline.</li>
	<li>Monitor the flow of injected FITC-dextran continuously for 20 min. On the FCFM-associated software, use the diameter button on the upper ruler in order to measure the blood vessels&#160;and&#160;categorize them as small (&#60;15 &#181;m) or large (&#62;15 &#181;m).</li>
	<li>Euthanize the animal with anesthesia overdose.</li>
</ol>

<ol><li>Chow, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anthracyclines+cause+endothelial+injury+in+pediatric+cancer+patients%3A+a+pilot+study%5BTitle%5D)+AND+%22J+Clin+Oncol%22%5BJournal%5D)">Anthracyclines cause endothelial injury in pediatric cancer patients: a pilot study.</a> J Clin Oncol. 24 (6), 925-928 (2006).</li>
<li>Nuver, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+chemotherapy-induced+cardiovascular+changes+in+patients+with+testicular+cancer%5BTitle%5D)+AND+%22J+Clin+Oncol%22%5BJournal%5D)">Acute chemotherapy-induced cardiovascular changes in patients with testicular cancer.</a> J Clin Oncol. 23 (36), 9130-9137 (2005).</li>
<li>Vos, F. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cell+effects+of+cytotoxics%3A+balance+between+desired+and+unwanted+effects%5BTitle%5D)+AND+%22Cancer+Treat+Rev%22%5BJournal%5D)">Endothelial cell effects of cytotoxics: balance between desired and unwanted effects.</a> Cancer Treat Rev. 30 (6), 495-513 (2004).</li>
<li>Kerbel, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%27Accidental%27+anti-angiogenic+drugs.+anti-oncogene+directed+signal+transduction+inhibitors+and+conventional+chemotherapeutic+agents+as+examples.Eur%5BTitle%5D)+AND+%22J+Cancer%22%5BJournal%5D)">'Accidental' anti-angiogenic drugs. anti-oncogene directed signal transduction inhibitors and conventional chemotherapeutic agents as examples.Eur.</a> J Cancer. 36 (10), 1248-1257 (2000).</li>
<li>Soultati, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+vascular+toxicity+from+chemotherapeutic+agents%3A+preclinical+evidence+and+clinical+implications%5BTitle%5D)+AND+%22Cancer+Treat+Rev%22%5BJournal%5D)">Endothelial vascular toxicity from chemotherapeutic agents: preclinical evidence and clinical implications.</a> Cancer Treat Rev. 38 (5), 473-483 (2012).</li>
<li>Tempelhoff, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood+coagulation+during+adjuvant+epirubicin%2Fcyclophosphamide+chemotherapy+in+patients+with+primary+operable+breast+cancer%5BTitle%5D)+AND+%22J+Clin+Oncol%22%5BJournal%5D)">Blood coagulation during adjuvant epirubicin/cyclophosphamide chemotherapy in patients with primary operable breast cancer.</a> J Clin Oncol. 14 (9), 2560-2568 (1996).</li>
<li>Ben Aharon, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxorubicin-induced+vascular+toxicity--targeting+potential+pathways+may+reduce+procoagulant+activity%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Doxorubicin-induced vascular toxicity--targeting potential pathways may reduce procoagulant activity.</a> PLoS One. 8 (9), e7515(2013).</li>
<li>Laemmel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibered+confocal+fluorescence+microscopy+%28Cell-viZio%29+facilitates+extended+imaging+in+the+field+of+microcirculation.+A+comparison+with+intravital+microscopy%5BTitle%5D)+AND+%22J+Vasc+Res%22%5BJournal%5D)">Fibered confocal fluorescence microscopy (Cell-viZio) facilitates extended imaging in the field of microcirculation. A comparison with intravital microscopy.</a> J Vasc Res. 41 (5), 400-411 (2004).</li>
<li>Al-Gubory, K. H., Houdebine, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+green+fluorescent+protein-expressing+cells+in+transgenic+animals+using+fibred+confocal+fluorescence+microscopy%5BTitle%5D)+AND+%22Eur+J+Cell+Biol%22%5BJournal%5D)">In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy.</a> Eur J Cell Biol. 85 (8), 837-845 (2006).</li>
<li>Bar-Joseph, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+bioimaging+as+a+novel+strategy+to+detect+doxorubicin-induced+damage+to+gonadal+blood+vessels%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">In vivo bioimaging as a novel strategy to detect doxorubicin-induced damage to gonadal blood vessels.</a> PLoS One. 6 (9), e23492(2011).</li>
<li>Kaushal, V., Kaushal, G. P., Mehta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+toxicity+of+anthracyclines+on+cultured+endothelial+cells%5BTitle%5D)+AND+%22Endothelium%22%5BJournal%5D)">Differential toxicity of anthracyclines on cultured endothelial cells.</a> Endothelium. 11 (5-6), 253-258 (2004).</li>
<li>Kim, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxorubicin-induced+platelet+cytotoxicity%3A+a+new+contributory+factor+for+doxorubicin-mediated+thrombocytopenia%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">Doxorubicin-induced platelet cytotoxicity: a new contributory factor for doxorubicin-mediated thrombocytopenia.</a> J Thromb Haemost. 7 (7), 1172-1183 (2009).</li>
<li>Walsh, J., Wheeler, H. R., Geczy, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+tissue+factor+on+human+monocytes+by+cisplatin+and+adriamycin%5BTitle%5D)+AND+%22Br+J+Haematol%22%5BJournal%5D)">Modulation of tissue factor on human monocytes by cisplatin and adriamycin.</a> Br J Haematol. 81 (4), 480-488 (1992).</li>
<li>Kotamraju, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxorubicin-induced+apoptosis+in+endothelial+cells+and+cardiomyocytes+is+ameliorated+by+nitrone+spin+traps+and+ebselen.+Role+of+reactive+oxygen+and+nitrogen+species%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species.</a> J Biol Chem. 275 (43), 33585-33592 (2000).</li>
<li>Vasquez-Vivar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+nitric+oxide+synthase-dependent+superoxide+generation+from+adriamycin%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Endothelial nitric oxide synthase-dependent superoxide generation from adriamycin.</a> Biochemistry. 36 (38), 11293-11297 (1997).</li>
</ol>

Evaluating chemotherapy-induced vascular toxicity is challenging due to the difficulty in visualizing the dynamics of vasculature in response to a stimuli in real-time. Numerous clinical studies have implicated that several chemotherapies cause direct vascular injury, yet the mechanism and characteristics of this toxicity remains to be elucidated. We have established a real-time, in vivo molecular imaging platform for evaluating the potential vascular toxicity of chemotherapy in mice comprising of fibered confoc...

Clinical evidence indicates that several classes of chemotherapies elicit a variety of vascular pathologies manifested by Raynaud phenomenon, hypertension, myocardialinfarction, cerebrovascular attack, and hepatic veno-occlusivedisease1,2. &#8220;&#8217;Accidental&#8217; anti-angiogenic drugs&#8221; is a fairly new term, which describes conventional chemotherapeutic agents that act as possible angiogenesis inhibitors, although they not originally developed for this purpose3-5 but designed to eliminate tumor cells by imposing as little &#8220;collateral damage&#8221; to normal cells as possible3. Several chemotherapies have been implied as vasculo-toxicants as observed in clinical studies using serum biomarkers. Among these are alkylating agents (such as cyclophosphamide), platinum compounds (such as cisplatin) and anthracyclines1,2,5-7.
Acute cardiovascular complications may occur as a result of vascular toxicity induced by chemotherapy. They may progress into chronic conditions like atherosclerosis and account for increased risk of late vascular morbidity. Yet, despite mounting clinical evidence, there is a paucity of designated studies emphasizing the mechanism of vascular toxicity and therefore, further elucidation of the exact pathogenesis they inflict is warranted.
A major challenge in revealing the mechanism of chemotherapy-induced vascular toxicity derives from the complexity of investigating vascular function in vivo. We describe herein a platform of high-resolution in vivo molecular imaging in mice that enables to capture blood flow and vessels&#8217; characteristics. This platform facilitates the detection of direct treatment-induced vascular effects: in real-time, as well as following them over a period of time within the same individuals.

<table><tbody><tr><td>general anesthesia</td><td>Fort Dodge Animal Health, IA, USA and Biove Laboratories, France</td><td></td><td>100 mg/kg ketaset and 6 mg/kg XYL-M2</td></tr><tr><td>depilatory cream (Veet)</td><td>ReckittBenckiser, Bristol, UK</td><td></td><td></td></tr><tr><td>30 G, 1/2 inch needle attached to 1 ml syringe</td><td></td><td></td><td></td></tr><tr><td>FITC dextran (10 mg/ml; MW 2,000 kDa)</td><td>Sigma</td><td>FD2000S</td><td>100 &amp;mu;l volume</td></tr><tr><td>Doxorubicin</td><td>Teva, Israel</td><td></td><td>8 mg/kg, Adriamycin</td></tr><tr><td>paclitaxel</td><td>Taro, Israel</td><td></td><td>1.2 mg/kg, Medexel</td></tr><tr><td>saline</td><td></td><td></td><td></td></tr></tbody></table>

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

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

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

In vivo continuous imaging at real-time
The imaging apparatus used here is a high definition, fibered confocal microscope, equipped with a probe that enables visualization of vasculature and its response to various stimuli as chemotherapy. This method is minimally invasive since although it may facilitate imaging of deep vessels or organ, it requires a small incision for the probe. The probe bundles consist of tens of thousands of fibers, microscope optics and a proprietary precision con...

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.

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

The overall goal of this procedure is to use a high definition fibered confocal microscope to visualize the vasculature and its response to various stimuli. This is accomplished by first calibrating the microscope and then injecting the animal intravenously with fitzy dextrin to facilitate visualization of the blood vessels, the vessels are then imaged with the fibroid confocal fluorescence microscope in real time, during and after the experimental treatment. Ultimately, this in vivo molecular imaging platform may be used for evaluating the potential vascular toxicity of agents such as chemotherapeutics in an animal model.Generally, individuals new to this method may struggle because setting up a system and capturing a stable image can be challenging. Demonstrating the procedure will be Dr.Sef, a postdoc from the laboratory. Before preparing the mouse for imaging, turn on the fiber confocal fluorescence microscope, and connect the mini zero 30 micro probe.Calibrate the device according to the manufacturer's instructions. When the microscope is ready, place the mouse in a supine position on a polystyrene stage, and then secure the mouse to the pad with surgical tape. Then incise the

chemotherapy-induced-vascular-toxicity-real-time-vivo-imaging-vessel

Research

JoVE Journal

Medicine

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Contrast Enhanced Vessel Imaging using MicroCT

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

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.

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

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

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.

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

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Learn fibered confocal fluorescent microscopy for real-time vascular imaging in live animal models. This method enables evaluation of chemotherapy-induced vascular toxicity using fluorescent dextrin visualization.

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

Explore More Videos