The effect of axotomy on firing and ultrastructure of the crayfish mechanoreceptor neurons and satellite glial cells.

Neurotrauma is among main causes of human disability and death. We studied effects of axotomy on ultrastructure and neuronal activity of a simple model object - an isolated crayfish stretch receptor that consists of single mechanoreceptor neurons (MRN) enwrapped by multilayer glial envelope. After isolation, MRN regularly fired until spontaneous activity cessation. Axotomy did not change significantly MRN spike amplitude and firing rate. However, the duration of neuron activity from MRN isolation to its spontaneous cessation decreased in axotomized MRN relative to intact neuron. [Ca2+] in MRN axon and soma increased 3-10 min after axotomy. Ca2+ entry through ion channels in the axolemma accelerated axotomy-stimulated firing cessation. MRN incubation with Ca2+ionophore ionomycin accelerated MRN inactivation, whereas Ca2+-channel blocker Cd2+ prolonged firing. Activity duration of either intact, or axotomized MRN did not change in the presence of ryanodine or dantrolene, inhibitors of ryanodin-sensitive Ca2+ channels in endoplasmic reticulum. Thapsigargin, inhibitor of endoplasmic reticulum Ca2+-ATPase, or its activator ochratoxin were ineffective. Ultrastructural study showed that the defect in the axon transected by thin scissors is sealed by fused axolemma, glial and collagen layers. Only the 30-50 µm long segment completely lost microtubules and contained swelled mitochondria. The microtubular bundle remained undamaged at 300 µm away from the axotomy site. However, mitochondria within the 200-300 µm segment were strongly condensed and lost matrix and cristae. Glial and collagen layers exhibited greater damage. Swelling and edema of glial layers, collagen disorganization and rupture occurred within this segment. Thus, axotomy stronger damages glia/collagen envelope, axonal microtubules and mitochondria.

Involvement of MAPK, Akt/GSK-3ß and AMPK/mTOR signaling pathways in protection of remote glial cells from axotomy-induced necrosis and apoptosis in the isolated crayfish stretch receptor.

Severe mechanical nerve injury such as axotomy can lead to neuron degeneration and death of surrounding glial cells. We showed that axotomy not only mechanically injures glial cells at the cutting location, but also induces necrosis or apoptosis of satellite glial cells remote from the transection site. Therefore, axon integrity is necessary for survival of surrounding glial cells. We used the crayfish stretch receptor that consists of a single mechanoreceptor neuron enveloped by satellite glial cells as a simple, but informative model object in the study of the role of various signaling proteins in axotomy-induced death of remote glial cells. After axon transection, stretch receptors were isolated and incubated in saline in the presence or without specific inhibitors of various signaling proteins. Inhibition of MEK1/2, p38, Akt, GSK-3ß and mTOR increased axotomy-induced apoptosis of remote glial cells, whereas inhibition of ERK1/2 and GSK-3ß enhanced necrosis. This suggests the involvement of these signaling proteins in protective, antiapoptotic and antinecrotic processes in the remote satellite glia surrounding the axotomized mechanoreceptor neuron.

Photodynamic effect of Radachlorin on nerve and glial cells.

BACKGROUND: Radachlorin, a chlorine-derived photosensitizer, is used currently in photodynamic therapy (PDT) of skin cancer. In this work we studied Radachlorin-PDT effect on peripheral nerve and glial cells that are damaged along with tumor tissue. METHODS: We used simple model objects - a crayfish stretch receptor that consists of a single sensory neuron surrounded by glial cells and crayfish nerve cord consisting of nerve fibers and ganglia. Radachlorin absorption and emission spectra were registered using spectrophotometer and spectrofluorimeter. Radachlorin accumulation and intracellular localization were studied using the fluorescence microscope. Necrotic and apoptotic cells were visualized using propidium iodide and Hoechst 33342. Neuronal activity was registered using standard electrophysiological methods. RESULTS: Radachlorin absorption spectrum in the physiological van Harreveld saline (pH 7.3) contained maximums at 420 and 654nm. Its fluorescence band 620-700nm had a maximum at 664nm. In the crayfish stretch receptor Radachlorin localized predominantly to the glial envelope and penetrated slightly into the neuron body and axon. Radachlorin rapidly accumulated in the crayfish nerve cord tissue within 30min. Its elimination in the dye-free solution occurred slower: 11% loss for 2h. Radachlorin-PDT inactivated the neuron and induced necrosis of neurons and glial cells and glial apoptosis at concentrations as low as 10(-10)-10(-9)M. CONCLUSIONS: Radachlorin rapidly accumulates in the nervous tissue, mainly in glial cells, and demonstrates very high photodynamic efficacy that characterize it as a promising photosensitizer.