Retro-orbital injection of FITC-dextran combined with isolectin B4 in assessing the retinal neovascularization defect

Abstract

Background A reliable and effective method is required to deliver agent that can aid the in vivo imaging of retinal vessels. The aim of the present study was to evaluate retro-orbital (RO) injection of fluorescein-labeled isothiocyanate dextran (FITC-dextran) as a method of demonstrating retinal neovascularization (NV) and avascular areas in oxygen-induced retinopathy (OIR) mice. Methods Different concentrations of FITC-dextran were used to compare the efficacy of this agent in perfusing the retinal vessels. Hematoxylin–eosin (HE) staining was used to evaluate the safety of RO injection. The vitreous blood vessels and extent of NV were assessed in P17 OIR mice using FITC-dextran and compared with the corresponding measurements obtained following isolectin B4 staining or the combination of both methods. Results The fluorescence of small vessels and neovascular tufts could be observed clearly following RO injection of 0.05 ml of 25 mg/ml or 50 mg/ml FITC-dextran. No visible damage to tissues adjacent to the injection site was discovered. Vitreous blood flow was gradually reduced from P0 to P5 and eventually disappeared in P17 OIR mice, as demonstrated by FITC-dextran perfusion. The retinal NV areas assessed by isolectin B4 were larger than those assessed by FITC-dextran, but the retinal avascular areas were smaller. The combination of both methods could conduce to distinguish non-functional blood vessels. Conclusions RO injection of FITC-dextran combined with isolectin B4 is an effective, optimal method for assessing the NV area and avascular area.

Morphine pharmacokinetics and opioid transporter expression at the blood-retina barrier of male and female mice

“…Opioid pharmacokinetics in the CNS may not only depend on the activity of transporter proteins but on the integrity of blood-CNS barriers. Fluorescence imaging of FITC or FITC-dextran extravasation is a common assay for measuring BBB or BRB permeability ( Li et al, 2011 ; Miyata and Morita, 2011 ; Liang et al, 2012 ; Frahm and Tobet, 2015 ; Natarajan et al, 2017 ; Xu et al, 2019 ). There have been reports of morphine exposure impairing barrier integrity in vitro through a reduction of tight junction protein ZO-1 ( Wen et al, 2011 ) as well as reports that chronic morphine exposure (via time-released pelleted implants) increases BBB permeability to fluorescently-labeled dextrans in mice ( Leibrand et al, 2019 ).…”

Protective effects of nattokinase against microvasculopathy and neuroinflammation in diabetic retinopathy

“…The periorbital area of the right eye was gently exposed. FITC‐dextran (50 mg/mL in 0.05 mL, molecular weight: 2000; Sigma‐Aldrich) was injected into the mouse's orbital venous sinus using a 27‐gauge needle with a 1 mL syringe 27 . The eyeballs were enucleated at 5 min after injection, and the whole‐mount retina was carefully removed for confocal imaging.…”

“…FITC‐dextran (50 mg/mL in 0.05 mL, molecular weight: 2000; Sigma‐Aldrich) was injected into the mouse's orbital venous sinus using a 27‐gauge needle with a 1 mL syringe. 27 The eyeballs were enucleated at 5 min after injection, and the whole‐mount retina was carefully removed for confocal imaging. The vascular leakage of retinal microvessels was calculated as the percentage of leakage area to the total retina area ( n = 6 retinas in each group).…”

Tight junction component protein claudin-1 deficiency in retinal pigment epithelium leads to early and intermediate age-related macular degeneration phenotypes in mice

“…After general anesthesia by MMB, mice were perfused with 0.2mg 70,000MW FITC-dextran per body weight (g) via the right retro-orbital injections. The injection method was followed as in the previous literatures (Li et al, 2011; Yardeni et al, 2011). After 5 min reflux, the mice were euthanized.…”