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Blake Hugo Fortes, Javier Nahmias, Manuel Tapia, Tsung-Han Chou, Giovanni Luca Romano, Vittorio Porciatti, Luis E Vazquez; Detection of dynamic changes in retinal vessel caliber in vivo using steady-state fluorescein angiography. Invest. Ophthalmol. Vis. Sci. 2017;58(8):738.
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© ARVO (1962-2015); The Authors (2016-present)
Decreased ocular perfusion is hypothesized to contribute to retinal ganglion cell death in glaucoma. To study factors that regulate ocular perfusion, we developed a method to measure in vivo changes in retinal vessel caliber in response to oxygen and carbogen.
Fluorescein 100 mg/ml solution was injected intraperitoneally (IP) in 6-month old C57/B6J mice. Mice were subsequently anesthetized and imaged 15 minutes after IP injection with a Heidelberg Retinal Angiography confocal scanning laser ophthalmoscope, a 55° lens and a sensitivity set to 60. After baseline imaging, mice were ventilated for 10 minutes with 100% oxygen, or carbogen (30% CO2, 70% O2), followed by room air. Repeat imaging using the same settings was acquired at 1 and 10 minutes of gas inhalation and 2 minutes after return to room air. Average diameter of 8 separate 0.5mm-long retinal vessel segments from different vessels were measured using ImageJ software (NIH). Vessel segments selected had ~80% pixel brightness saturation in the baseline image. Average diameter in the same segments after treatment were compared to baseline using a paired t test. N refers to the number of experiments using different mice on different days; p<.05 was considered significant.
The retinal vasculature was easily visualized with IP injection of 10µl-100 µl fluorescein. 40 µl fluorescein had the most stable fluorescence brightness over time; steady-state brightness was observed at 15 minutes and lasted up to 30 minutes after IP injection. 100% oxygen resulted in a reduction in retinal vessel caliber (0.71-fold of baseline; N=4, p<0.0001) at 1 minute after inhalation. Conversely, carbogen resulted in an increase in retinal vessel caliber (1.23-fold of baseline; N=4, p<0.0001) at 1 minute after inhalation. Changes in caliber were observed throughout the entire vasculature, including large and small vessels, as well as arteries and veins, and persisted during the observation period of 10 minutes. Vessel caliber returned to baseline 2 minutes after returning to room air.
We report a useful method for detecting dynamic, bi-directional changes in mouse retinal vessel caliber in vivo. Data from a companion abstract (Nahmias, et al) suggests vessel caliber depends on retinal vascular smooth muscle tone. Co-measurement of blood flow velocity is required to estimate dynamic changes in ocular perfusion in future studies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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