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

Summary

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.

Abstract

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. Yet, albeit 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 to evaluate the potential acute vascular toxicity of anti-cancer therapies.

We have set up a platform of in vivo, high-resolution molecular imaging in mice, 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.

Introduction

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-occlusivedisease^1,2. “’Accidental’ anti-angiogenic drugs” is a fairly new term, which describes conventional chemotherapeutic agents that act as possible angiogenesis inhibitors, although they not originally developed for this purpose^3-5 but designed to eliminate tumor cells by imposing as little “collateral damage” 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 anthracyclines^1,2,5-7.

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’ 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.

Protocol

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; 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

1. Turn the device ON.
2. Connect the microprobe (mini0/30).
3. Calibrate the device according to manufacturer’s instructions.

2. Mice Preparation for Imaging 

1. Anesthetize by a subcutaneous injection of both Ketaset (100 mg/kg) and XYL-M2 (6 mg/kg). Confirm proper anesthetization by unresponsiveness to toe-pinch.
2. Incise the skin below the groin in order to reveal the femoral arterial vessels. Keep the incision site moist with saline following incision.
3. 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 the 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 µl; 10 mg/ml; 2,000 kDa) facilitates visualizing the femoral microvasculature by FCFM. Doxorubicin (100 µl; 8 mg/kg, Adriamycin) or saline will also be later administered IV into the pre-heated tail vein.
4. Position the mouse supinely on a polystyrene stage. Secure the mouse to the pad and maintain position using surgical duct tape.

3. Imaging of Femoral Blood Vessels by FCFM During and After Administration of Doxorubicin 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).

1. Perform all time-laps analyses using the LSU 488 nm wavelength laser. 
   NOTE: The main unit detector detects the filtered (500 - 650 nm) emitted fluorescence. The acquired images are reconstructed afterwards and displayed at a rate of 12 frames/sec.
2. Disconnect carefully the syringe from the needle and attach a new syringe containing FITC dextran. Administer (IV) 100 µl of FITC dextran.
3. Shift 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.
4. 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 µl doxorubicin or saline.
5. 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 and categorize them as small (<15 µm) or large (>15 µm).
6. Euthanize the animal with anesthesia overdose.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
general anesthesia	Fort Dodge Animal Health, IA, USA and Biove Laboratories, France		100 mg/kg ketaset and 6 mg/kg XYL-M2
depilatory cream (Veet)	ReckittBenckiser, Bristol, UK
30 G, 1/2 inch needle attached to 1 ml syringe
FITC dextran (10 mg/ml; MW 2,000 kDa)	Sigma	FD2000S	100 μl volume
Doxorubicin	Teva, Israel		8 mg/kg, Adriamycin
paclitaxel	Taro, Israel		1.2 mg/kg, Medexel
saline