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Updated: Sep 4, 2025
Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images
Published on: May 19, 2023
In-vivo assessment of retinal vessel diameters and observer variability in mice: A methodological approach
Lukas Streese1, Jeannine Liffert1,2, Walthard Vilser3
1Department of Sport, Exercise and Health, Medical Faculty, University of Basel, Basel, Switzerland.
Insights
Using fluorescein contrast enhancement significantly reduces variability in measuring central retinal arteriolar (CRAE) and venular (CRVE) equivalents in mice. This method improves precision for retinal vessel analysis in rodent models, aiding translation to human studies.
Area of Science:
- Ophthalmology
- Cardiovascular Research
- Animal Models
Background:
- Central retinal arteriolar (CRAE) and venular (CRVE) diameter equivalents are established predictors of cardiovascular and all-cause mortality in humans.
- Assessing CRAE and CRVE in mice is crucial for preclinical research, but variability in measurements can hinder translation to clinical findings.
Purpose of the Study:
- To evaluate the impact of fluorescein contrast enhancement on inter- and intraobserver variability in measuring CRAE and CRVE in mice.
- To compare the precision of fluorescein-enhanced imaging versus non-enhanced imaging for retinal vessel analysis in a rodent model.
Main Methods:
- Retinal images (with and without fluorescein) from C57BL mice were analyzed by two independent investigators to assess interobserver variability.
- One investigator re-analyzed images to determine intraobserver variability.
- Time-course imaging after fluorescein injection was performed to characterize vessel response.
Main Results:
- Fluorescein enhancement significantly reduced interobserver variability for both CRAE (r=0.99 vs. r=0.65) and CRVE (r=0.99 vs. r=0.79) compared to non-enhanced images.
- Intraobserver variability was also lower with fluorescein for CRAE (r=0.99 vs. r=0.48) and CRVE (r=0.98 vs. r=0.86).
- Fluorescein injection created stable vascular staining, ideal for precise measurements.
Conclusions:
- Fluorescein contrast enhancement optimizes measurement variability in mouse retinal vessel analysis.
- Standardizing image acquisition timing post-fluorescein injection is recommended.
- This rodent model approach can bridge the gap between animal research and human clinical studies.
Background:
Central retinal arteriolar (CRAE) and venular (CRVE) diameter equivalents are predictive for cardiovascular and all-cause mortality in humans. The aim of this study was to investigate the inter- and intraobserver variability for the assessment of CRAE and CRVE in mice using fluorescein contrast enhancement as compared to crude analysis.
Methods:
Three high quality images with (F) and without fluorescein (NF) of eight mice (type C57BL) were recorded and analysed by two independent experienced investigators to investigate interobserver variability. In addition, one investigator analysed 20 F and 20 NF images twice to investigate intraobserver variability. The time course of CRAE and CRVE vessel responses after fluorescein injection were recorded in one mouse every 30 seconds for 15 minutes.
Results:
The interobserver variability was lower in F images compared to NF images for CRAE (r = 0.99, p < 0.001 vs. r = 0.65, p = 0.083) and CRVE (r = 0.99, p < 0.001 vs. r = 0.79, p = 0.019). Intraobserver variability for CRAE (r = 0.99, p < 0.001 vs. r = 0.48, p = 0.032) and CRVE (r = 0.98, p < 0.001 vs. r = 0.86, p < 0.001) were lower in F compared to NF images. Fluorescein injection induced vascular staining mimicking vessel dilation (+14%) followed by a long-lasting stable staining phase well suited for precise measurements.
Conclusions:
Measurement variability can be optimized by use of fluorescein as contrast enhancement in mice. Standardization for time of image acquisition after fluorescein injection is advisable. Translation of static retinal vessel analysis into a rodent model has the potential to bridge the research gap between proof of concept studies in animals and clinical studies in humans.
