Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment
January 7th, 2015
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.
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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Overview
This article describes the method of fibered confocal fluorescent microscopy (FCFM) for imaging physiological phenomena at cellular and sub-cellular levels in animal subjects. The technique allows for real-time visualization of vasculature and its responses to various stimuli.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Imaging Techniques
Background
- Fibered confocal microscopy provides high-definition imaging capabilities.
- It is particularly useful for studying vascular responses in live animal models.
- Challenges exist in setting up the system and capturing stable images.
- Fitzy dextrin is used to enhance visualization of blood vessels.
Purpose of Study
- To visualize the vasculature and its response to stimuli in real-time.
- To evaluate potential vascular toxicity of agents like chemotherapeutics.
- To demonstrate the FCFM technique for researchers new to the method.
Methods Used
- Calibration of the fibered confocal microscope.
- Intravenous injection of fitzy dextrin into the animal.
- Real-time imaging of blood vessels during and after treatment.
- Demonstration of the procedure by a trained postdoc.
Main Results
- Successful visualization of vasculature in live subjects.
- Real-time imaging allows for assessment of vascular responses.
- Potential applications in evaluating vascular toxicity of drugs.
- Identification of challenges faced by new users of the technique.
Conclusions
- FCFM is a valuable tool for in vivo molecular imaging.
- It enhances understanding of physiological responses at the cellular level.
- Further training and practice are recommended for effective use.
Frequently Asked Questions
Executive Industry Relevance
Real-time in vivo imaging of vascular responses enables early detection of chemotherapy-induced vascular toxicity, a critical factor in preclinical safety assessment. This approach supports mechanistic de-risking by distinguishing vascular from cardiotoxic mechanisms, informing go/no-go decisions in oncology pipeline prioritization. The platform provides quantitative, reproducible vascular metrics that enhance predictive confidence in lead identification stages.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of vascular toxicity mechanisms independent of direct myocyte injury, clarifying target-related vascular liabilities.
- Operational Value: Provides real-time, quantitative readouts of vessel impairment for hypothesis testing in discovery biology.
Screening & Assay Development
- Scientific Value: Generates standardized, high-resolution vascular imaging data suitable for assay development and compound screening.
- Operational Value: Supports reproducible visualization of blood vessel dynamics across test conditions, enabling reliable compound evaluation.
Translational & Preclinical Research
- Scientific Value: Bridges discovery findings to preclinical validation by modeling acute vascular events that may precede end-organ injury.
- Operational Value: Facilitates longitudinal, within-subject vascular monitoring using each animal as its own control, reducing variability.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from early target validation through lead identification to preclinical safety assessment, providing real-time vascular response data at each stage.
- Discovery Biology: Supports hypothesis testing of vascular toxicity mechanisms and pathway clarification in vivo.
- Screening: Enables assay-ready visualization of vascular responses with quantitative diameter and permeability measurements.
- Analytics: Delivers real-time, sub-cellular vascular metrics including vasoconstriction onset and leakage quantification.
- Translational Research: Connects acute vascular imaging findings to preclinical models of vascular morbidity and cardiovascular risk.
- Enterprise Reuse: Establishes a reusable imaging platform for vascular toxicity profiling across multiple therapeutic modalities.
Operational & Enterprise Impact
- Scientific Value: Mechanistic de-risking of vascular toxicity through direct visualization of vessel impairment dynamics.
- Operational Value: Standardized, reproducible imaging workflow with internal controls and real-time monitoring.
- Strategic Value: Informs early go/no-go decisions by identifying vascular liabilities before significant investment.
- Portfolio Impact: Enables risk-adjusted prioritization of candidates based on vascular safety profiles.
Implementation Considerations
- Expertise in intravital microscopy and surgical vessel exposure is required for reliable data acquisition.
- Instrumentation includes fibered confocal fluorescence microscope, microprobe, and laser scanning unit with 488 nm wavelength capability.
- Cross-team standardization depends on consistent probe stabilization, injection techniques, and image analysis protocols.
- Adaptation considerations include vessel size thresholds (<15 μm) and timing of measurements relative to compound administration.
- Practical limitations include challenges in maintaining probe stability during animal respiration and injection procedures.
