OBJECTIVE: The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high-throughput, and cost-effective alternative technique that minimizes animal usage. METHODS: Sodium fluorescein fundus angiography was performed in non-diabetic and diabetic Brown Norway rats on days 0, 7, 14, 21, and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post-study eyes were fixed, dissected, and stained (isolectin B4) to measure required parameters for permeability quantification including total vessel length per retinal volume, radius, and thickness. RESULTS: In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from days 14-28 (P < .01, P < .001, P < .0001). CONCLUSIONS: This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive, and cost-effective alternative to the Evans blue assay.
OBJECTIVE: The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high-throughput, and cost-effective alternative technique that minimizes animal usage. METHODS:Sodium fluorescein fundus angiography was performed in non-diabetic and diabetic Brown Norway rats on days 0, 7, 14, 21, and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post-study eyes were fixed, dissected, and stained (isolectin B4) to measure required parameters for permeability quantification including total vessel length per retinal volume, radius, and thickness. RESULTS: In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from days 14-28 (P < .01, P < .001, P < .0001). CONCLUSIONS: This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive, and cost-effective alternative to the Evans blue assay.
Authors: Claire L Allen; Katarzyna Wolanska; Naseeb K Malhi; Andrew V Benest; Mark E Wood; Winfried Amoaku; Roberta Torregrossa; Matthew Whiteman; David O Bates; Jacqueline L Whatmore Journal: Front Cell Dev Biol Date: 2021-09-07
Authors: Naseeb K Malhi; Claire L Allen; Elizabeth Stewart; Katherine L Horton; Federica Riu; Jennifer Batson; Winfried Amoaku; Jonathan C Morris; Kenton P Arkill; David O Bates Journal: Am J Physiol Heart Circ Physiol Date: 2022-03-18 Impact factor: 5.125
Authors: Marlene E Da Vitoria Lobo; Nick Weir; Lydia Hardowar; Yara Al Ojaimi; Ryan Madden; Alex Gibson; Samuel M Bestall; Masanori Hirashima; Chris B Schaffer; Lucy F Donaldson; David O Bates; Richard Philip Hulse Journal: Pain Date: 2022-03-29 Impact factor: 7.926