Background: The photodynamic therapy using 5-aminolevulinic acid is one of the new therapeutic modalities for malignant glioma yet. There has been a controversy as to the mechanism of cell damage in acute phase induced by 5-ALA-mediated PDT. In this study, acute morphological and histological sequelae of 5-ALA-mediated PDT in the C6 spheroid model were examined and the cell damage mechanism in acute phase was discussed.
Methods: Various sizes of C6 spheroids were incubated for 4 h in 100 microg/ml of 5-ALA and subsequent by irradiated with a diode laser at various total energies (635+/-5 nm, 5-100 mW/cm2, total light dose 2.5-50 J/cm2). For investigating morphological changes, the spheroid's diameter was measured just before and after PDT. Hematoxylin-eosin and TUNEL assays were performed on cryosections of the spheroids after PDT as a histological assessment. Fluorescence microscopic examination was performed using annexin V-FITC and propidium iodide (PI) to distinguish necrosis and apoptosis. Control groups with laser only, ALA only or no treatment were used in comparison.
Results: All spheroids with 5-ALA-mediated PDT enlarged in their diameters immediately after PDT. They increased light transparency in superficial zone, which indicated cell membrane damage. There were no significant differences in the expansion of spheroids of a same size among the total light doses at 2.5-50 J/cm2. Large spheroids were less expanded by PDT at total light dose 25 J/cm2 as compared with small ones (P<0.01). H.E. staining showed condensed nucleus and cytoplasm in the superficial layer. However, these cells were negative for TUNEL staining. The spheroid after 5-ALA-mediated PDT was apparently densely positive for PI staining. Double stains for PI and annexin V-FITC indicated that positive cells for annexin V-FITC were also positive for PI. Only annexin V-FITC-positive cells were scarcely demonstrated. These findings were not seen in any of control groups.
Conclusions: PDT-mediated 5-ALA for experimental glioma using spheroid model in the present in vitro study resulted in rapid and significant cells damage, which indicated acute necrosis just after PDT.