Animals
Table 1 shows information regarding strain, age, sex and supplier for the animals used in the different experiments throughout the study.
Table 1
Information regarding strain, age, sex and supplier for the animals used in the different experiments throughout the study.
Model/experiment | Strain | Supplier | Age at start of experiment | Sex |
70 KDa Fluorescein, FITC-dextran IVT/dose response | C57Bl/6J | Charles River laboratories (Germany) | 10–12 weeks old | Female |
Endotoxin-induced uveitis (LPS IVT) | C57Bl/6J | Charles River laboratories (Germany) | 12 weeks old | Female |
STZ diabetes | C57Bl/6J | Charles River laboratories (France) | 10 weeks old | Male |
Spontaneous CNV/anti-VEGF | JR5558 | Charles River laboratories outsourcing (Germany) | 8 and 13 weeks old | Male and female |
FITC = Fluorescein isothiocyanate; IVT = intravitreal; CNV = Choroidal Neovascularisation; VEGF = Vascular Endothelial Growth factor |
All animals received food and water ad libitum, in a 12-hour day/night cycle, temperature-controlled environment. Animal experiments were approved by the Federal Food Safety and Veterinary Office of Switzerland (references BS-2730 and BS-2734) and conducted in strict adherence to the Swiss federal ordinance on animal protection and welfare, as well as according to the rules of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research guidelines, and in compliance with the ARRIVE guidelines.
For intravitreal (IVT) lipopolysaccharide (LPS) injections, animals were anaesthetized using isoflurane 3%, O2 100%. For all other experiments mice were anaesthetized using the subcutaneous combination of medetomidine (Dorbene®, 0.5 mg/kg, Graeub AG, Bern, Switzerland), midazolam (5 mg/kg, Roche Pharma AG, Grenzach-Whylen, Germany) and fentanyl (Curamed®, 0.05 mg/kg, Actavis Switzerland AG, Regensdorf, Switzerland). For recovery experiments, mice were then antagonized with a combination of buprenorphin (Bupaq®, 0.2 mg/kg, Streuli Pharma AG, Uznach, Switzerland), atipemazol (Alzane®, 2.5 mg/kg, Graeub AG, Bern, Switzerland) and flumazenil (Anexate®, 0.5 mg/kg, Roche Pharma AG, Grenzach-Whylen, Germany).
Endotoxin-induced Uveitis
Prior to IVT application, a solution of LPS (Cat L6529; Sigma, St Gallen, Switzerland) was prepared in sterile Dulbecco’s Phosphate Buffered Saline (DPBS; Life technologies, Switzerland) from frozen aliquots stored at -20°C with a final concentration of 1 ng/1 µl. Animals were anesthetized with isoflurane, and drops of local anesthetic (Novesin®; OmniVision, Switzerland) and mydriatic (Tropicamide 0.5% SDU Faure, Théa Pharma S.A., Schaffhausen, Switzerland)) applied. The eyes were rinsed with povidone iodine 5% (iso-Betadine® 5%, MEDA-Pharma GmbH & Co.KG, Bad Hombourg, Germany), ), followed by sterile PBS, then with a Nanofil Syringe and connected 34 gauge needle (NF34BL-2) (both World Precision Instruments International, Friedberg, Germany) 1 µl/eye LPS or PBS (control) was injected. Finally, animals were placed back into the housing box for recovery.
After 24 hours, mice were anaesthetized using subcutaneous anaesthesia, then fluorescein angiography (FA), and ocular coherence tomography (OCT) images of the retina and vitreous were taken to confirm the presence of infiltrating inflammatory cells. Following imaging, mice underwent fluorophotometry.
Stz Diabetes
Animals were divided in two groups, receiving either streptozotocin (STZ; 50 mg/kg body weight, 7.5 mg/ml, prepared in 0.05 M sodium citrate buffer, pH 4.5) or PBS injected intraperitoneally (IP) for five consecutive days. STZ was prepared and stored in a refrigerator at least 30 min before administration. Five days after the last STZ or PBS doses, blood glucose was measured via tail vein puncture using an AlphaTrak blood glucose monitoring system (Abbott Laboratories Inc USA, Alameda,CA) and test strips (Alpha Trak2, Zoetis Schweiz, Zürich Switzerland). Development of diabetes was defined as a blood glucose levels higher group than 14 mmol/L (250 mg/dl). Animals with glucose levels lower than 250 mg/dl were not reinjected or included in the study. The animal’s weight and glucose were monitored throughout the study, and only mice with continuously elevated blood glucose levels were considered as diabetic. For fluorophotometry measurements, animals were measured at 10 weeks post-STZ.
Jr5558 Spontaneous Cnv Mice
Male and female JR5558 mice 33 were used throughout the studies. For the time course and anti-VEGF pharmacological treatment studies, animals were 13, and 8 weeks old, respectively. For drug treatment experiments, mice underwent baseline fluorescein angiography (FA) 34 and fluorophotometry assessment on week 8 on the day prior to antibody dosing, to assign animals to treatment groups with statistically equal (P > 0.05; unpaired t-test) numbers of lesions and fluorescein optical density measurements in different eye compartments. Anti-VEGF (B20-4.1 36) or IgG control antibodies were given I.P. one day after baseline, and then a repeat dose 3 days later (1ml/100g body weight, 2 doses in total). FA and fluorophotometry data were collected again one week after the initial antibody dose, to compare pre- and post-treatment effects of anti-VEGF and IgG control. For baseline and post-treatment assessment of JR5558 mice, the 45-minute time point was selected for fluorophotometry.
Fluorophotometry
Fluorophotometry measurements were carried out on a scanning ocular fluorophotometer (Fluorotron Master Research Mouse Edition; OcuMetrics, Inc., Mountain View, CA).
To ensure that fluorescence values measured for fluorescein in tissue and plasma were in the linear range, we ran standard curves using PBS and plasma (used in experiments for correction to circulating levels). In both cases, linear relationships were observed (R2 > 0.99; Fig. 1).
For in vivo experiments, animals were anaesthetized using subcutaneous anaesthesia. Following anaesthesia eyes were dilated with 0.5% tropicamide (Tropicamide 0.5% SDU Faure, Théa Pharma, Schaffhausen, Switzerland), then 5 minutes later fluorescein solution (cat 46960; Sigma-Aldrich, St Gallen, Switzerland) or FITC-dextran 70kDa (cat 46945; Sigma-Aldrich, St Gallen, Switzerland) was administered either IV, IVT or SC, at doses described in the results sections. For most consistent results, we observed it was best to administer fluorescein following anaesthesia.
After fluorescein injection, 3.2-mm plano contact lenses (Cantor and Nissel, Northamptonshire, UK) were placed on the mouse eyes. Contact lenses are used to prevent drying, which can lead to cataract formation. The animals were then placed on a temperature controlled (37°C) stage, and the eyes aligned parallel to the optic device. Eye-scans were recorded from both left and right eyes using 450–490 nm excitation and 520–600 nm emission detection, at time points indicated in each result section. Eye scans took approximately 20 seconds per scan.
In some cases following fluorophotometry, blood samples (25 µl) were taken from the tail vein for plasma preparation (Microvette CB 300K2E; Sarstedt AG, Germany) to normalize scans to circulating fluorescein, to account for potential administration differences. Blood samples centrifuging at 10,000 x g for 10 minutes, then 10 µl of the resulting plasma was diluted in 990 µl PBS in a microcuvette protected from light (PS Micro Photometer Cuvette 2ml; LP Italiana, Milan, Italy) and scanned on the Fluorotron machine using the provided cuvette holder, also using 450–490 nm excitation and 520–600 nm emission detection.
To quantify Fluorotron measurements, raw data was exported as txt. files, then plotted using Microsoft Excel. The average area under the curve (AUC) was calculated for 5 steps in each of the regions of the scans corresponding to retina, vitreous and anterior chamber peaks, as determined by eye compartment experiments (Fig. 2). Data not normalised to plasma fluorescein are expressed as mean optical density (OD) ± SEM.
For plasma normalisation, circulating fluorescein was calculated by averaging the maximum peak values of duplicate scans. Fluorescein levels in eye compartments were then expressed as a ratio to plasma fluorescein, by dividing raw AUC averages by plasma values. Data are expressed as mean ± SEM OD ratio of ocular compartment:plasma fluorescein.
Statistics
Raw data were exported from the fluorophotometer in .txt files and copied into Microsoft Excel (Microsoft Corporation, Redmond, WA). Processing and analysis were carried out using Excel and GraphPad Prism 6 (GraphPad Software, La Jolla, CA) software. For statistical comparison of only two groups, an unpaired t-test was employed. One-way ANOVA followed by Newman Keul’s multiple comparisons test was used for comparisons of 3 or more groups. For time course data, Two-way Analysis of Variance ANOVA (alpha 0.05) with repeated measures, followed by multiple comparison tests were used (see Figure legends for further details). Outliers were removed prior to statistical analysis using Chauvenet's criteria, with outliers defined as being ± 2 × standard deviations of mean. Data are presented as Mean ± SEM with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.