2.3. Vascular Permeability Assay with FITC-Dextran

Microscopic observations were made up to 48 h after MRT with video documentation. For the assay, we used FITC-dextran of MW 2 × 10⁶ Daltons, (Fluorescein isothiocyanate from Sigma, Taufkirchen, Germany). FITC-dextran has a Stroke Ratio of 270 Angstrom [50], which converts to approximately 27 nm.

We also employed red fluorescent polystyrene microspheres (FluoSpheres^TM Carboxylate-Modified Microspheres, Cat #: F8810, ThermoFisher, Waltham, MA, USA). The microspheres have a diameter of 100 nm with a coefficient variation in size of 5% [51]. Their surfaces are pre-coated with a high density of carboxylic acids, which endows the microspheres with a highly charged and relatively hydrophilic surface layer. The surface charges range between 0.1 and 2.0 mEq/g, which makes them stable to a relatively high concentration of electrolytes (max. 1 M univalent salt) and prevents agglomerates [52]. We selected this microsphere because the highly charged surface reduces their attraction to cells, which makes them ideal for studying vascular permeability.

CAM: CAMs were either left untreated or intravenously injected with FITC-dextran and red fluorescent microspheres. Moreover, we applied 1.0 µg of recombinant VEGF-A165 protein (Peprotech, London, UK) on the surface of 7 CAMs, and compared them against VEGF-untreated CAMs. A semi-quantitative analysis of the extravasated FITC-dextran was performed in vivo in the CAM. The blood flow and FITC-dextran extravasation were monitored every 15 min. The intensity of perivascular FITC-dextran was evaluated according to the score presented in the caption of Figure 1. The video sequences documented the presence and site of the microbeam stripes, the optically empty zones, the damaged medium- and large-sized vessels, the damage to the capillary network, and extravasation of the fluorescent probes.

Images from intravital microscopy showing increased vascular permeability in CAM after exposure to MRT peak doses of 75 Gy; (a,b): normal CAM vasculature; (c–e): vasculature after VEGF treatment. Note: (a) forty-five minutes after exposure to MRT, the vascular permeability is increased, as demonstrated by the extravasation of FITC-dextran (green-fluorescent halos around the blood vessels). Conversely, in (b), the microspheres did not diffuse into the surrounding tissue but remained affixed as red-fluorescent dots along the microbeams path. Left side: (c) at the site of VEGF application (asterisks indicate the edge of the Thermanox^® coverslip), the vascular permeability increased as early as 10 min after MRT, as shown by the halos of extravasated FITC-dextran (some marked by arrows). Right side: in the non-treated zone, no such signs of increased vascular permeability were observed simultaneously. Images (d,e): parts of (c) at higher magnification. Image (f): schematic representation of the vascular permeability window after 75 Gy of MRT. The score: (0) = no FITC extravasation; (1) = small non-confluent FITC “halo” surrounding the capillaries; (2) = FITC “halos” start merging but they are not completely confluent; (3) = the “halos” are completely confluent. In (a–e), black stripes on the radiochromic film indicate the path of the microbeams.

Glioblastoma Xenograft: Forty-five minutes after irradiation, we performed a vascular permeability assay in a selected group of mice; MRT + Cisplatin (n = 4), MRT alone (n = 7), Cisplatin alone (n = 4), and control (n = 4). A solution of 3% FITC-dextran in sterile saline was injected (0.3 mL) into the tail vein of mice. In all cases, tumors were harvested approximately 30 min after injection of the fluorescent compound and fixed in 2% paraformaldehyde.