Ringer lactate solution

Erythrocytes were obtained from the French blood national agency (EFS-LR agreement number # 2018001378). Blood was collected in EDTA tubes (BD vacutainer^®, Franklin Lakes, NJ, USA). Erythrocytes were washed 3 times with an isotonic Ringer-lactate solution (B. Braun, Melsungen, Germany) and prepared at 20% hematocrit in phosphate buffered saline solution/5 mM glucose (PBS/0.1% glucose). Glycation was induced by incubating erythrocytes in the absence (G0) or presence of D-glucose at three increasing concentrations (5 (G5), 50 (G50) and 100 mM (G100)). A previous study, performed in our laboratory, showed that such in vitro conditions represent a suitable erythrocyte glycation model, inducing HbA1c percentages similar to those that can be measured in diabetic patients [16 (link)]. After 5 days of incubation at 37 °C, erythrocytes were washed 3 times with Ringer-lactate solution before a direct use by FACS, ektacytometry and for endothelial cells stimulation. Quantitative determination of hemoglobin in erythrocytes was performed with Drabkin’s colorimetric assay.

We primed micro-osmotic Alzet pumps (model 1007D) under sterile conditions one day before implantation and left them in an incubator at 37.0°C overnight. We anesthetized the animals with a ketamine (Ketavet, Pfizer, Germany, 150 mg/kg) and xylazine (Rompun, Bayer, Germany, 15 mg/kg) mixture dissolved in 8.5 ml of sterile 0.9% saline. Head skin incision was performed exposing the skull.
We implanted the cannula 0.2 mm to the right from Bregma corresponding to the location of the right ventricle. A drop of cyanoacrylate clay (Weicon, Germany) was introduced between the cannula cap and the skull. We slowly moved the cannula down until the cap touched the skull. We left the animals for 5 min to ensure drying of the clay. Then, we carefully sutured the skin above the cannula with a 5–0 polypropilen thread (Ethicon, USA).
Animals received lidocaine gel (Xylocain, AstraZeneca, Germany) locally and 0.5 ml of ringer lactate solution (B Braun Melsungen, Germany) intraperitoneally to substitute the liquid loss. We placed the cage with operated animals onto the 37°C warm bed for 2 hours.
The animals received a total amount of 84±16.8 µl of PEDF (20 µg/ml in CSF) or CSF at a pumping rate of 0.5±0.1 µl/hour.