PDT and vascular response

The vascular response to PDT was investigated intra-vitally using the skin-fold chamber model and ex-vivo in skin samples harvested 24 hours after PDT.

The PDT induced vascular damage was histologically investigated in SKH1-hr mice by collecting the skin in the illuminated area and of the contralateral side at day 1 after PDT (group 6, Table 1). The PDT illumination was performed as described above for the visual skin damage experiment. Skin samples were harvested 24 hours after PDT under 2–3% Isoflurane in oxygen anaesthesia before mice were sacrificed. Skin samples were snap frozen and stored at -80°C. For the ex-vivo mouse skin experiments, extra pieces of skin was harvested from the control mice and immediately used for that experiment (group 3, Table 1). Frozen skin was imbedded in Tissue Tek O.C.T.^™ Compound (Sakura Holland B.V.) and 5 and 50 μm cross-sections were cut with a cryostat, mounted on glass slides (StarFrost, Waldemar Knittel Glasbearbeitungs) and air-dried. The 50 μm sections were used for fluorescence labelled IHC staining of CD31 and CD144 on the same day and the staining procedure was performed as described previously [27].

In a last group of SKH1-hr mice the vascular response to ALA-PDT and BF-200 PDT was investigated at depth in skin using the chamber model and intra-vital confocal microscopy (group 7, Table 1). Confocal images of the subcutaneous musculature and lower dermis were recorded before and after illumination using a Zeiss Laser Scanner Microscope 510 now equipped with a 10x Plan-Neofluar objective, heated stage and a gas anaesthesia supply unit. All measurements and PDT illumination was performed under 2–3% Isoflurane in oxygen anaesthesia.

PDT was performed using a 630 nm laser (Visuals 630, Carl Zeiss B.V. Sliedrecht, NL) and a microlens (Medlight SA, Ecublens, Switzerland). The total light dose and irradiance (100 J/cm² at 50 mw/cm²) used in previous experiments with 514 nm were translated to 630 nm based on the in-vivo absorption spectrum of PpIX in mouse skin and the photon energy as described above. This resulted in a light dose of 130.6 Jcm^-2 at 65.3 mWcm^-2 either delivered in a single illumination or according to a light fractionation scheme. In this skin-fold window experiment photobleaching during PDT could not be monitored and the illumination of the first light fraction was standardized to be 6.53 Jcm^-2, which is consistent with 5 Jcm^-2 at 514 nm. The transmission images were also used to determine the vascular area pre and post PDT. The area of a vessel was measured by drawing regions of interest in the LSM aim software. While the animal is repositioned between measurements care was taken to measure the same length of vessel for each series of images pre and post PDT.