Imaging and Histopathological Analysis of Microvascular Angiogenesis in Photodynamic Therapy for Oral Cancer

Abstract

The objective of this study is to use imaging and histopathological analysis to characterize and monitor microvascular responses to photodynamic therapy (PDT). In vivo chicken chorioallantoic membranes (CAMs) and a stimulated malignant oral lesions animal model were used to determine the blood flow and the biological activities of Photofrin^® (2.5 mg/kg) exposed to different laser power densities at 630 nm. The vascular changes, the velocity of the blood flow, the speckle flow index (SFI) of fluorescence changes, and ultrastructure damage in the microvasculature before and after PDT were recorded. The subcellular localization of Photofrin^® revealed satisfactory uptake throughout the cytoplasm of human red blood cells at 10 s and 20 s before PDT. The mean blood-flow velocities of the veins and arteries were 500 ± 40 and 1500 ± 100 μm/s, respectively. A significant decrease in the velocities of the blood flow in the veins and arteries was detected in the CAM model after PDT. The veins and arteries of CAMs, exposed to the power densities of 80, 100, and 120 mW/cm², had average blood-flow velocities of 100 ± 20, 60 ± 10, and 0 μm/s and 300 ± 50, 150 ± 30, and 0 μm/s, respectively. In the stimulated malignant oral lesions animal model, the treated tumors exhibited hemorrhage and red blood cell extravasation after PDT. The oxyhemoglobin and total hemoglobin levels decreased, which resulted in a decrease in tissue oxygen saturation, while the deoxyhemoglobin levels increased. PDT using Photofrin^® has the ability to cause the destruction of the targeted microvasculature under nonthermal mechanisms selectively.

Keywords: chicken chorioallantoic membrane; photodynamic therapy; stimulated malignant oral lesions.

Figure 1

Under the fluorescent microscope (40×), the subcellular localization of Photofrin^® revealed satisfactory uptake throughout the cytoplasm of CAM RBCs at 10 s and 20 s (A,B).

Figure 2

The PDT-induced vascular damage was evaluated, and observable damage with vascular constriction or occlusion was evaluated under a stereomicroscope. The area where the blood vessel color turns white represents the vascular damage induced by PDT (right-hand side). In contrast, the area where the blood vessel color remains unchanged as a control represents the vascular tissue not damaged by PDT (left-hand side).

Figure 3

An example of stimulated squamous cell carcinoma on the buccal mucosa in image of OCT concept (A), under white light (B), and 3 h after intravenous application of Photofrin^® during PDT of tumor (C); the tumor area is marked with the symbol (*).

Figure 4

The histopathological findings and fluorescent images (100×) of stimulated squamous cell carcinoma on the buccal mucosa of hamsters (A,B) were identified. Severe internal hemorrhage and red blood cell extravasation with the destruction of the tumor 12 h after PDT were demonstrated (C,D) (200×).