29th Annual Meeting of the American Society of Photobiology

Downtown Marriot

Chicago, Il.

PDT-induced Death of Isolated Neuronal and Glial Cells

Abstract-
In order to study PDT-induced cell death in a model organ consisting of several tightly interacting cellular types we photosensitized isolated crayfish stretch receptor (consisting of receptor muscle and sensory neuron surrounded by Schwann cells) with sulphonated alumophthalocyanine Photosens (0.1 or 10 M). After 30-min incubation with Photosens the preparation was irradiated with He-Ne laser (632.8 nm; 0.3 W/cm²) until irreversible firing abolition. Then, in 1-2; 3-6, or 10-20 h cells were fixed with glutaraldehyde, washed, fluorochromed with Hoechst 33342, and photographed. In some experiments non-fixed photosensitized cells were stained with propidium iodide in order to reveal impairment of the plasma membrane integrity leading to necrosis. Two distinct types of irreversible firing abolition were electrophysiologically recorded at high and low PDT intensities: firing acceleration followed by abrupt depolarization block, or gradual firing inhibition until irreversible cessation, respectively. Using different substrates or inhibitors it was shown that free radicals, inhibition of ATP production, and increase in cytosolic Ca²⁺ enhanced the efficiency of photodynamic cell killing. Apoptotic nucleus fragmentation was never observed in the sensor neuron under PDT impact, though both strong (10 M) and weak (0.1 M) photosensitization caused time-dependent nucleus shrinkage. Propidium iodide staining demonstrated necrotic neuron death in 15-30 min after photosensitization with 10 M Photosens and delayed necrosis occurring in 2-3 h after firing abolition in the case of photosensitization with 0.1 M Photosens. Proliferation, necrosis and apoptosis of glial cells were observed after functional inactivation of stretch receptor. At both PDT intensities apoptosis of some fraction of glial cells was most prominent in 3-6 h after neuron inactivation. It seems that neuron photoinactivation provokes prominent multimodal glial response along with direct photodynamic killing of glial cells. Supported by RFBR and Competition Center at SPbGU.

Keywords: PDT, cell death, neuron, glia