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Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats
Published on: December 5, 2017
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Tracking endothelium-dependent NO release in pressurized arteries.
Lillian Wallis1, Lucy Donovan1, Aaron Johnston1
1The Vascular Pharmacology Group, Department of Pharmacology, University of Oxford, Oxford, United Kingdom.
Frontiers in Physiology
|February 10, 2023
Summary
Researchers evaluated two nitric oxide (NO) fluorescent dyes in intact arteries, finding DAR-4M AM effective for measuring NO release from endothelial cells (ECs) in cardiovascular research.
Area of Science:
- Vascular Biology
- Cardiovascular Physiology
- Biomedical Optics
Background:
- Endothelial cell (EC) dysfunction, marked by reduced nitric oxide (NO) bioavailability, is an early indicator of cardiovascular disease.
- Current methods struggle to reliably assess NO levels in intact arteries, hindering cardiovascular disease research.
- Assessing NO bioavailability is crucial for understanding vascular tone regulation and disease progression.
Purpose of the Study:
- To compare the efficacy of two NO-sensitive fluorescent dyes, Cu2FL2E and DAR-4M AM, for measuring NO in intact arteries.
- To establish a reliable method for quantifying NO release from endothelial cells under physiological conditions.
- To investigate the potential of fluorescent dye-based NO detection in advancing cardiovascular research.
Main Methods:
- Confocal fluorescence microscopy was employed to evaluate Cu2FL2E and DAR-4M AM in cell-free systems and isolated rat mesenteric arteries.
- Pressure myography and en face imaging were used to visualize endothelial cells (ECs) and vascular smooth muscle cells (SMCs) in intact arteries.
- Fluorescence ratio (FR/FR0) using an elastin-labeled reference was calculated to correct for motion artifacts and quantify NO-dye accumulation over time.
Main Results:
- Both dyes showed increased fluorescence with NO donors in cell-free chambers.
- DAR-4M AM, but not Cu2FL2E, demonstrated a time-dependent increase in FR/FR0 in intact arteries upon stimulation with acetylcholine (ACh) or an NO donor (SNAP).
- L-NAME, an NO synthase inhibitor, blocked the ACh-induced fluorescence increase, confirming the specificity of DAR-4M AM for NO production by ECs.
Conclusions:
- DAR-4M AM, when combined with elastin labeling, provides a reliable method for measuring NO release from ECs in intact arteries.
- This technique advances the assessment of vascular NO bioavailability, crucial for understanding cardiovascular health and disease.
- Future studies can utilize this method to determine basal NO release from ECs, offering new insights into vascular function.
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