Photodynamic treatment that causes intense oxidative stress and cell death is currently used in neurooncology. However, along with tumor cells, it may damage healthy neurons and glia. In order to study photodynamic effect on normal nerve and glial cells, we used crayfish stretch receptor, a simple system consisting of only two identified sensory neurons surrounded by glial cells. Photodynamic treatment induced firing abolition and necrosis of neurons as well as necrosis and apoptosis of glial cells. Nerve growth factor but not brain-derived neurotrophic factor or epidermal growth factor protected glial cells but not neurons from photoinduced necrosis and apoptosis. Inhibitors of tyrosine kinases or protein kinase JNK eliminated anti-apoptotic effect of nerve growth factor in photosensitized glial cells but not neurons. Therefore, these signaling proteins were involved in the anti-apoptotic activity of nerve growth factor. These data indicate the possible presence of receptors capable of recognizing murine nerve growth factor in crayfish glial cells. Thus, intercellular signaling mediated by nerve-growth-factor-like neurotrophin, receptor tyrosine kinase, and JNK may be involved in crayfish glia protection from apoptosis induced by photodynamic treatment.

To investigate the mechanisms of oxidative injury of neurons and glia, we studied the photodynamic effect on isolated stretch receptor that consists of only two sensory neurons enwrapped by satellite glial cells. Photodynamic therapy (PDT), a potent inducer of oxidative stress, is a prospective method for destruction of brain tumors. PDT induced functional inactivation and necrosis of neurons, necrosis, apoptosis, and proliferation of glial cells. The roles of calmodulin, calmodulin-dependent kinase II, phospholipase C, protein kinases A and C, and phosphodiesterase in these processes were studied by using their inhibitors: fluphenazine, KN-93, D-609, H89, staurosporine, and papaverine, respectively. PDT-induced firing abolishment was enhanced by H89 or papaverine, whereas staurosporine acted oppositely. Fluphenazine or KN-93 reduced necrosis of neurons and glial cells. H89 enhanced necrosis of neurons, whereas staurosporine enhanced necrosis of glial cells. Inhibition of protein kinases A and C enhanced PDT-induced glial apoptosis. Photodynamic gliosis was prevented by KN-93 or staurosporine. These data indicate possible involvement of calmodulin and calmodulin-dependent kinase II in photoinduced necrosis of neurons and glia. Protein kinase C could protect glial cells from necrosis and apoptosis and participate in photoinduced gliosis and loss of neuronal activity. Protein kinase A maintained neuronal firing and protected neurons from photoinduced necrosis and glial cells from apoptosis. Phosphodiesterase reduced necrosis of photosensitized neurons and glia. Thus, Ca(2+)- and cAMP-mediated signaling pathways were involved in photooxidative injury of neurons and glia. Their pharmacological modulation may differently change the efficacy of photodynamic injury of neurons and glial cells.

Neurons and glial cells can protect each other from stress and following death by mutual exchange with neurotrophins. In order to examine involvement of different neurotrophic factors in neuroglial interactions in a photosensitized crayfish stretch receptor, a simple model object consisting of only two sensory neurons enveloped by glial cells, we studied the influence of glial cell line-derived neurotrophic factor (GDNF), neurturin, and ciliary neurotrophic factor (CNTF) on its photodynamic injury. Photodynamic treatment, which causes strong oxidative stress, induced firing abolition and necrosis of neurons, necrosis, and apoptosis of glial cells. GDNF significantly reduced photoinduced neuronal necrosis and neurturin but not CNTF showed a similar tendency. Both of them significantly reduced necrosis and apoptosis of glial cells. At the ultrastructural level, neurons and glial cells treated with GDNF in the darkness contained large mitochondria with well-developed cristae, numerous ribosomes, polysomes, rough endoplasmic reticulum (ER), and dictyosomes. This indicated the high level of bioenergetic, biosynthetic, and transport processes. Photodynamic treatment caused swelling and vacuolization of mitochondria, dictyosomes, and ER. It also impaired formation of glial protrusions and double membrane vesicles that transfer glial material into the neuron. GDNF prevented photoinduced mitochondria swelling that disturbed the cellular bioenergetics and cytoplasm vacuolization associated with injury of intracellular organelles. It also preserved the structures involved in protein synthesis and transport: rough ER, dictyosomes, polysomes, microtubule bundles, submembrane cisterns, and double membrane vesicles. GDNF-mediated maintenance of metabolism and ultrastructure of photosensitized neurons and glial cells may be the basis of its neuro- and glia protective effects.

The mechanisms of photodynamic (PD) damage to neurons and gliocytes are discussed. The spike reactions of neurons are described, with stimulation at high concentrations of photosensitizer and inhibition at low concentrations, accompanying necrosis. Glial cells developed both necrosis and apoptosis. Local laser inactivation of neurons increased light-induced apoptosis of gliocytes, i.e., neurons maintained gliocyte survival. Inter-and intracellular signaling plays an important role in the photolesioning of these cells. Studies using inhibitors and activators of signal proteins demonstrated the involvement of the Ca(2+)-dependent, adenylate cyclase, and tyrosine kinase pathways in the responses of neurons and gliocytes to PD treatment. Pharmacological modulation may alter the selectivity of PD neuron and gliocyte damage and the efficacy of PD treatment.

To study the involvement of neuroglial interactions in photodynamic damage of crayfish stretch receptor, which consists of only two neurons surrounded by satellite glial cells (SGCs), we attempted to proteolytically uncouple neurons and glia and then compare the responses of these cells to photosensitization when intercellular communications were intact or impaired. After incubation of isolated stretch receptors with pronase or collagenase they were photosensitized with Photosens, a mixture of sulfonated alumophthalocyanines AlPcS(n) (n = 2, 3, and 4; mean n = 3.1). In the next 6 hr the preparations were double fluorochromed with propidium iodide and Hoechst-33342 to visualize necrotic and apoptotic cells. Proteolytic treatment shortened bioelectric neuron response and precipitated its functional inactivation; however, it did not significantly impair neuron morphology and did not induce its necrosis either in the darkness or under photosensitization. Photodynamic treatment induced necrosis of neurons and SGC and apoptosis of glial cells. Pronase but not collagenase increased percent of necrotic and apoptotic SGCs in the darkness and thus reduced the number of glial cells around the neuron; however, both pronase and collagenase prevented photodynamically induced apoptosis of glial cells. The involvement of neuron-to-glia signaling interactions in this phenomenon is suggested.

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Photodynamic therapy (PDT) inhibited and irreversibly abolished firing, caused necrosis of neurons, necrosis, apoptosis and proliferation of glial cells in the isolated crayfish stretch receptor. The role in these processes of the central components of Ca 2+ -mediated signaling pathway: phospholipase C, calmodulin, calmodulin-dependent kinase II, and protein kinase C was studied using their inhibitors: ET-18, fluphenazine, KN-93, or staurosporine, respectively. ET-18 reduced functional inactivation of neurons, necrosis and apoptosis of glial cells. Fluphenazine and KN-93 reduced PDT-induced necrosis of neurons and glial cells. Staurosporine enhanced PDT-induced glial apoptosis. PDTinduced gliosis was prevented by KN-93 and staurosporine. Therefore, phospholipase C participated in neuron inactivation and glial necrosis and apoptosis. Calmodulin and calmodulin-dependent kinase II were involved in PDTinduced necrosis of neurons and glial cells but not in glial apoptosis. Protein kinase C protected glia from apoptosis and participated in PDT-induced gliosis and loss of neuronal activity. These data may be used for modulation of PDT of brain tumors.