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....

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.

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