A fluorescence-based method to assess plasma protein extravasation in rat dura mater using confocal laser scanning microscopy

Abstract

We describe a nonradioactive, fluorescence-based method to assess plasma protein extravasation (PPE) in rat dura mater using confocal laser scanning microscopy (CLSM). Unilateral PPE can be induced by electrical stimulation of the ipsilateral trigeminal ganglion (TG) and is widely used as an experimental migraine model. The gold standard to determine PPE in the meninges is based on the detection of radiolabeled albumin (^[125]I-BSA). The aim of this study was to develop a nonradioactive, histological method to quantify PPE in the meninges. The fluorescent dye Evans Blue (50 mg/kg) was injected intravenously to the rat 7 min prior to TG stimulation. PPE in dura mater was detected by a CLSM. The amount of extravasated Evans Blue in the dura mater was measured at six to eight regions of interest (ROIs) in the vicinity of large meningeal vessels. The ratio of the average fluorescence intensity within dura mater of the “stimulus side”, compared to the contralateral “control side”, was calculated for each animal. By using this method, The PPE ratio was 1.67±0.12 (n=5). Intravenous injection of three different dosages of the 5HT_1B/1D-receptor agonist sumatriptan (25, 50, and 100 μg/kg) 15 min prior to stimulation attenuated PPE by 42±12%, 49±9%, and 86±15%, respectively (p<0.01). The approximated ED₅₀ value was 48 μg/kg. Our results are in accordance with previous reports in the literature using the radioactive approach. We conclude that CLSM is a safe, sensitive, and reliable method to assess PPE in rat meninges in an experimental migaine model.