Investigating the Effects of Photodynamic Therapy on Cells of the Nervous System In Vitro

The effect of photodynamic therapy (PDT) on neural cells is important when tumours are adjacent to or within the nervous system. The primary purpose of this study was to investigate PDT treatments using the photosensitiser meta-(tetrahydroxyphenyl) chlorin (m-THPC) on cells of the peripheral nervous system. There is evidence that, during clinical PDT-treatment of tumour sites near important nerve structures, peripheral neural cells respond differently to m-THPC-mediated PDT than do tumour cells. In this study, the response to PDT-treatment of rat dorsal root ganglia (DRG) neurones co-cultured with satellite cells were compared with separately cultured human breast adenocarcinoma cell line (MCF-7) in innovative in vitro culture systems. Epi-fluorescence microscopy confirmed that m-THPC was incorporated into all cell types, excluding photosensitiser uptake as a reason for differences in the response to m-THPC-mediated PDT in vitro. Sensitivity of cells exposed to m-THPC- mediated PDT (0-10 ~g/ml m-THPC with 1 J/cm2white light) was determined in a 1 mm thin bespoke 3-dimensional (3D) collagen hydrogel culture system. Cell death was quantified using propidium iodide (PI) exclusion and DRG neurones were identified in co-culture using immunocytochemistry for β-tubulin. MCF-7 cells, DRG satellite cells and astrocytes (of the central nervous system) were significantly more sensitive to m-THPC-mediated PDT treatments than were DRG neurones. Importantly, the dose of interest, 4 f.lg/ml m-THPC-mediated PDT, caused no significant DRG neurone death in comparison with untreated controls, but was sufficient to elicit substantial cell death in the other cell types The cell death protocol was validated using a second photosensitiser, hypericin (0-3~g/ml with 1 J/cm2 white light), which caused substantial DRG neurone death equivalent to other cell types studied. Following m-THPC-mediated PDT treatments, ORG neurones displayed a loss of their neurites. An assessment of the ability of these cells to regenerate neurites after m-THPC-mediated PDT treatments was performed and neurite extension was found to be equivalent to that in untreated controls, demonstrating that the treated ORG neurones were viable and retained their neurite projecting function. Inhibiting specific cell antioxidant pathways gave an insight into the mechanism by which neurones survived m-THPC-mediated PDT. The results suggested that ORG neurones were protected from the phototoxic effects of m-THPC-mediated PDT by superoxide dismutase-1 (500-1) and the glutathione synthase antioxidant pathways. DRG neurones used in this study survived m-THPC-mediated PDT under conditions sufficient to kill tumour cells and other nervous system cells. Identifying and understanding this phenomenon could provide the basis for developing PDT treatments that reduce nerve damage during cancer therapy. The question of whether m-THPC-mediated PDT has different effects on ORG neurone survival and/or function depending on which part of the cell is illuminated is of particular interest. Preliminary experiments are described in which 2D and 3D tissue engineered nerve cultures were developed and assessed for use in evaluating focally applied m-THPC-mediated PDT treatment to different parts of DRG neurones. These peripheral nerve culture systems have controllable cellular environments that make them attractive experimental tools for further investigations into the effect of PDT on peripheral nerves.