During Photodynamic Therapy (PDT) of cancer, cells are killed by 1O2, which is generated in a photosensitized process. A photosensitizer (PS) is applied to the tissue and irradiated with light to form an exited molecule. This generates 1O2 from ground state oxygen, which then induce processes that result in cell death (e. g. apoptosis or necrosis). We have used patch-clamp recordings from cultured neurons to provide insight into the earliest events following the light induced perturbation. Hippocampal neurons from rat embryos were cultured using standard methods and used after 10-20 DIV. Incubation period was 5h in normal culture medium with the PS Pyropheophorbide-a (PPa), which has a high lipophilicity and is mainly located in the membrane. Laser depended detection of singlet oxygen was carried out according to Snyder et al. (Photochem. Photobiol. (2006), 82; 177-84). Irradiation and whole-cell electrophysiological recordings were performed in ABM. Relevant values for cellular response were membrane resistance (RM), capacitance (CM) and holding current (IHold), as well as the firing activity. The I/V relationship was investigated with 1s duration current steps of 0.1nA from -0.4 to 1nA. Accumulation of PPa as imaged by fluorescence was shown to be correlated to the generation of 1O2. Nevertheless, the membrane parameters remained unchanged in the presence of the sensitizer and absence of light. During irradiation, RM (down), CM (up) and IHold (more negativ) changed immediately only in treated cells. The spiking pattern was altered significantly, reflected by changes in spike threshold, frequency and tendency for fast APs to be followed by a plateau phase. These effects were correlated with the intensity and/or duration of illumination. Since we have previously documented that the lifetime and diffusion range of singlet oxygen are very small, its effects will be restricted. The intra-membrane generated 1O2 alters the composition of the lipid and also proteins or channels. This is reflected both in the immediate response to low irradiation-dose and in formation of long plateaus, which could be caused by different influences on K+ and Ca2+ channels.
Neurology; Photodynamic Therapy; Singlet Oxygen; Electophysiology; Photosensitizing Agents