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.

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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>
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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 skin below the groin of a seven to eight week old female ICR mouse to reveal the femoral arterial vessels, keeping the incision site moist with saline.Use a red lamp light to heat the tail for approximately 10 seconds when the tail veins have dilated, insert a 27 gauge and a half inch needle attached to a one milliliter syringe filled with saline into one of the veins. Inject saline to ensure that the vein is open. Then carefully replace the syringe of saline with a syringe containing 10 milligrams per milliliter, Fitz Dextrin.Next, inject 100 microliters of the Fitz Dextrin into the tail vein, and attach a syringe containing eight milligrams per kilogram of doxorubicin to the needle. Then shift the mini zero 30 micro probe to a suitable field of view following adjustment to the Z axis. Fix the probe over the incision site.The trickiest parts of the procedure are finding a suitable field of view and keeping the probe in place to ensure that both the animal and the probe aren't moving during the injection or become loose due to the animal's breathing. We secure the probe with a lot of tape. Now wait for the initial signal to fade until a clear and focused signal is visible.Then after recording the baseline blood flow for a short, roughly 32nd stabilization period, administer 100 microliters of the treatment through the tail vein shunt. Monitor the flow of the injected fit dextrin continuously for 10 minutes with the laser scanning unit set to the 488 nanometer wavelength. Finally, in the fibroid confocal fluorescence microscope associated software, use the diameter button on the upper ruler to measure the blood vessels.Doxorubicin induces a rapid vasoconstriction of small vessels less than 15 micrometers in diameter within two to five minutes of treatment with a complete disappearance of the Fitz Dextrin fluorescence by eight minutes. In this representative experiment, some animals exhibited a reduction of the fluorescent signal in the blood vessel, and an increase in the signal in the area surrounding the vessels within the perivascular region. A few seconds after doxorubicin administration in these mice, administration of the high molecular weight dextrin induced an increase in the vessel permeability, resulting in a leakage of the signal from the blood vessel into the surrounding tissue.Once mastered, this technique can be completed in minutes if it is performed properly.

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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

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