Protection of the Crayfish Mechanoreceptor Neuron and Glial Cells from Photooxidative Injury by Modulators of Diverse Signal Transduction Pathways.

Oxidative stress is the reason of diverse neuropathological processes. Photodynamic therapy (PDT), an effective inducer of oxidative stress, is used for cancer treatment, including brain tumors. We studied the role of various signaling pathways in photodynamic injury and protection of single neurons and satellite glial cells in the isolated crayfish mechanoreceptor. It was photosensitized with alumophthalocyanine Photosens in the presence of inhibitors or activators of various signaling proteins. PDT eliminated neuronal activity and killed neurons and glial cells. Inhibitory analysis showed the involvement of protein kinases Akt, glycogen synthase kinase-3ß (GSK-3ß), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinases 1 and 2 (MEK1/2), calmodulin, calmodulin-dependent kinase II (CaMKII), adenylate cyclase, and nuclear factor NF-κB in PDT-induced necrosis of neurons. Nitric oxide (NO) and glial cell-derived neurotrophic factor (GDNF) reduced neuronal necrosis. In glial cells, protein kinases Akt, calmodulin, and CaMKII; protein kinases C and G, adenylate cyclase, and p38; and nuclear transcription factor NF-κB also mediated PDT-induced necrosis. In contrast, NO and neurotrophic factors nerve growth factor (NGF) and GDNF demonstrated anti-necrotic activity. Phospholipase Cγ, protein kinase C, GSK-3ß, mTOR, NF-κB, mitochondrial permeability transition pores, and NO synthase mediated PDT-induced apoptosis of glial cells, whereas protein kinase A, tyrosine phosphatases, and neurotrophic factors NGF, GDNF, and neurturin were involved in protecting glial cells from photoinduced apoptosis. Signaling pathways that control cell survival and death differed in neurons and glia. Inhibitors or activators of some signaling pathways may be used as potential protectors of neurons and glia from photooxidative stress and following death.

Epigenetic regulation of death of crayfish glial cells but not neurons induced by photodynamic impact.

Epigenetic processes are involved in regulation of cell functions and survival, but their role in responses of neurons and glial cells to oxidative injury is insufficiently explored. Here, we studied the role of DNA methylation and histone deacetylation in reactions of neurons and surrounding glial cells to photodynamic treatment that induces oxidative stress and cell death. Isolated crayfish stretch receptor consisting of a single mechanoreceptor neuron surrounded by glial cells was photosensitized with aluminum phthalocyanine Photosens that induced neuron inactivation, necrosis of the neuron and glia, and glial apoptosis. Inhibitors of DNA methylation 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine) reduced the level of PDT-induced necrosis of glial cells but not neurons by 1.3 and 2.0 times, respectively, and did not significantly influence apoptosis of glial cells. Histone deacetylase inhibitors valproic acid and trichostatin A inhibited PDT-induced both necrosis and apoptosis of satellite glial cells but not neurons by 1.6-2.7 times. Thus, in the crayfish stretch receptor DNA methylation and histone deacetylation are involved in epigenetic control of glial but not neuronal necrosis. Histone deacetylation also participates in glial apoptosis.

Intense focused ultrasound as a potential research tool for the quantification of diurnal inflammatory pain.

Quantifying pain through assay of a human's or animal's response to a known stimulus as a function of time of day is a critical means of advancing chronotherapeutic pain management. Current methods for quantifying pain, even in the context of etiologies involving deep tissue, generally involve stimulation by quantifiable means of either cutaneous (heat-lamp tests, electrical stimuli) or both cutaneous and subcutaneous tissue (von Frey hairs, tourniquets, etc.) or study of proxies for pain (such as stress, via assay of cortisol levels). In this study, we evaluate the usefulness of intense focused ultrasound (iFU), already shown to generate sensations and other biological effects deep to the skin, as a means of quantifying deep diurnal pain using a standard animal model of inflammation. Beginning 5 days after injection of Complete Freund's Adjuvant into the plantar surface of the rat's right hind paw to induce inflammation, the rats were divided into two groups, the light-phase test group (09:00-18:00h) and the dark-phase test group (23:00-06:00h), both of which underwent iFU application deep to the skin. We used two classes of iFU protocol, motivated by the extant literature. One consisted of a single pulse (SP) lasting 0.375s. The other, a multiple pulse (MP) protocol, consisted of multiple iFU pulses each of length 0.075s spaced 0.075s apart. We found the night group's threshold for reliable paw withdrawal to be significantly higher than that of the day group as assayed by each iFU protocol. These results are consistent with the observation that the response to mechanical stimuli by humans and rodents display diurnal variations, as well as the ability of iFU to generate sensations via mechanical stimulation. Since iFU can provide a consistent method to quantify pain from deep, inflamed tissue, it may represent a useful adjunct to those studying diurnal pain associated with deep tissue as well as chronotherapeutics targeting that pain.

Protection effect of GDNF and neurturin on photosensitized crayfish 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.

Intense focused ultrasound can reliably induce sensations in human test subjects in a manner correlated with the density of their mechanoreceptors.

Sensations generated by intense focused ultrasound (iFU) can occur cutaneously and/or at depth, in contrast to other forms of stimulation (e.g., heat, electricity), whose action usually occurs only at the skin surface, or mechanical stimulation (e.g., von Frey hairs, calibrated forceps, tourniquets) that compress and thus stimulate all tissue. Previous work on iFU stimulation has led to the hypothesis that the tactile basis of iFU stimulation should correlate with the density of mechanoreceptors at the site of iFU stimulation. Here we tested that hypothesis, correlating a "two-point" neurological examination-a standard measure of superficial mechanoreceptor density-with the intensity of superficially applied iFU necessary to generate sensations with high sensitivity and specificity. We applied iFU at 1.1 MHz for 0.1 s to the fingertip pads of 17 test subjects in a blinded fashion and escalated intensities until they consistently observed iFU-induced sensations. Most test subjects achieved high values of sensitivity and specificity, doing so at values of spatially and temporally averaged intensity measuring <100 W/cm(2). Moreover, the test subjects' sensitivity to iFU stimulation correlated with the density of mechanoreceptors as determined by a standard two-point discrimination neurological examination, consistent with earlier hypotheses.

On the role of phosphatidylinositol 3-kinase, protein kinase b/Akt, and glycogen synthase kinase-3ß in photodynamic injury of crayfish neurons and glial cells.

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. To study the involvement of signaling processes in photodynamic injury or protection of neurons and glia, we used crayfish mechanoreceptor consisting of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens. Application of specific inhibitors showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3ß participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3ß pathway was not involved as a whole in photodynamic injury of crayfish neurons and glia but its components, Akt and glycogen synthase kinase-3ß, independently and cell specifically regulated death of neurons and glial cells. According to these data, necrosis in this system was a controlled but not a non-regulated cell death mode. The obtained results may be used for the search of pharmacological agents selectively modulating death and survival of normal neurons and glial cells during photodynamic therapy of brain tumors.

Elevated activity of the crayfish stretch receptor neuron increases resistance of surrounding glial cells to apoptosis induced by photodynamic treatment.

Neuroglial interaction is very important for functioning and survival of nerve and glial cells. In the present work, we studied the influence of the intense neuronal activity on survival of the isolated crayfish stretch receptor neuron and surrounding glial cells subjected to photodynamic treatment, which induces intense oxidative stress. In the experimental group, neurons were stimulated by multiple extensions of the receptor muscle for 1h so that the firing rate did not fall below 10-15 Hz, whereas in the control group, the receptor muscles were relaxed and neurons were silent. After stimulation, the preparations were photosensitized with alumophthalocyanine Photosens and irradiated by 670 nm laser diode. The isolated stretch receptors were stained with propidium iodide and Hoechst 33342, which reveal the nuclei of the necrotic and the apoptotic cells, respectively. The level of apoptosis of photosensitized glial cells was significantly lower in the experimental group compared to the resting control. Necrosis of neurons and glial cells was not significantly influenced. Therefore, elevated neuronal activity increased the resistance of the surrounding glial cells to photoinduced apoptosis. This could be attributed to the depletion of the energetic resources, which are transferred from glia into the neuron to support its firing, or to the neurotrophic neuron-to-glia signaling.

Protection of crayfish glial cells but not neurons from photodynamic injury by nerve growth factor.

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.

Crayfish mechanoreceptor neuron prevents photoinduced apoptosis of satellite glial cells.

Interactions between neurons and glia play a key role in the development, functioning and survival of the nervous system. However, the influence of neurons on glial cells has received less attention than the role of glia in supporting neural functions. We here investigated the role of isolated crayfish stretch receptor neuron in the death of satellite glial cells under photodynamic impact. After staining with aluminum phthalocyanine photosens, the neuronal cell body was locally irradiated with a focused beam of He-Ne (633 nm, 200 W/cm2) or semiconductor laser (650 nm, 50 W/cm2). This rapidly abolished neuronal activity. The whole preparation was then subjected to total laser irradiation with lower intensity (633 nm, 0.3 W/cm2), which induced death of glial cells. Double staining of the preparation with propidium iodide and Hoechst 33342 in the following 6-7h allowed the visualization of necrotic, apoptotic and alive cells. Previous neuron inactivation with the focused laser beam was found to increase photodynamically-induced apoptosis but not necrosis of satellite glial cells enwrapping the axon. Therefore, the intact neuronal cell body protected satellite glial cells against photoinduced apoptosis. Altogether the data indicate that mechanoreceptor neurons release some signaling molecules involved in the prevention of glial apoptosis. This may provide integrity of the stretch receptor organ and its resistance to injurious factors.

Magnetic micro- and nanoparticle mediated activation of mechanosensitive ion channels.

Most cells are known to respond to mechanical cues, which initiate biochemical signalling pathways and play a role in cell membrane electrodynamics. These cues can be transduced either via direct activation of mechanosensitive (MS) ion channels or through deformation of the cell membrane and cytoskeleton. Investigation of the function and role of these ion channels is a fertile area of research and studies aimed at characterizing and understanding the mechanoactive regions of these channels and how they interact with the cytoskeleton are fundamental to discovering the specific role that mechanical cues play in cells. In this review, we will focus on novel techniques, which use magnetic micro- and nanoparticles coupled to external applied magnetic fields for activating and investigating MS ion channels and cytoskeletal mechanics.

In vivo analysis of proprioceptive coding and its antidromic modulation in the freely behaving crayfish.

Although sensory nerves in vitro are known to convey both orthodromic (sensory) and antidromic (putatively modulating) action potentials, in most cases very little is known about their bidirectional characteristics in intact animals. Here, we have investigated both the sensory coding properties and antidromic discharges that occur during real walking in the freely behaving crayfish. The activity of the sensory nerve innervating the proprioceptor CBCO, a chordotonal organ that monitors both angular movement and position of the coxo-basipodite (CB) joint, which is implicated in vertical leg movements, was recorded chronically along with the electromyographic activity of the muscles that control CB joint movements. Two wire electrodes placed on the sensory nerve were used to discriminate orthodromic from antidromic action potentials and thus allowed for analysis of both sensory coding and antidromic discharges. A distinction is proposed between 3 main classes of sensory neuron, according to their firing in relation to levator muscle activity during free walking. In parallel, we describe 2 types of antidromic activity: one produced exclusively during motor activity and a second produced both during and in the absence of motor activity. A negative correlation was found between the activity of sensory neurons in each of the 3 classes and identified antidromic discharges during walking. Finally, a state-dependent plasticity of CBCO nerve activity has been found by which the distribution of sensory orthodromic and antidromic activity changes with the physiological state of the biomechanical apparatus.

Fully tuneable stochastic resonance in cutaneous receptors.

Stochastic resonance describes a phenomenon whereby the addition of "noise" to the input of a nonlinear system can improve sensitivity. "Fully tuneable stochastic resonance" is a particular form of the phenomenon that requires the matching of two time scales: one being that of the subthreshold periodic stimulus of the system and the other being the noise-induced response of the system. First proposed in 1981, stochastic resonance has been reported in a wide range of biological systems; however, conclusive experimental evidence for fully tuneable stochastic resonance in biological systems is limited. Evidence of fully tuneable stochastic resonance in the response of slowly adapting type I mechanoreceptors in the toad is presented. The results are extended to include the first evidence supporting fully tuneable stochastic resonance in psychophysical experiments, namely tactile detection thresholds, indicating that the human CNS is capable of accessing the improved information available via fully tuneable stochastic resonance.

Effects of gadolinium and tetrodotoxin on the response of slowly adapting type I mechanoreceptors to mechanical stimulation in frog dorsal skin.

To elucidate the excitatory mechanism of mechanoreceptors innervating the frog skin, we examined the effects of gadolinium (Gd3+) and tetrodotoxin (TTX) on the response of single-unit activity of slowly adapting type I mechanoreceptors to mechanical stimulation topically applied to the receptive field (RF). Recordings were made from 46 fibers responding to mechanical stimulation with von Frey hairs, which caused an irregular firing pattern with slow adaptation. Application of a mechanically gated channel blocker, Gd3+ (30 microM), and a Na+ channel blocker, TTX (3 microM), caused the suppression of discharge rates, which was characterized by the conversion of a slowly adapting to a rapidly adapting discharge pattern. The administration of a high-voltage-activated (HVA) Ca2+ channel blocker, Cd2+ (100 microm), inhibited the unit discharge and caused the conversion of a slowly adapting to a rapidly adapting discharge pattern. Tonic discharges evoked by anodal electrical stimulation were inhibited by the application of Gd3+ or TTX. Electron microscopic examination showed that the cytoplasm of Merkel cells seen in the RF contained numerous Merkel granules. These results suggest that the excitatory mechanism of frog cutaneous mechanoreceptors may be mediated by the activation of Gd(3+)-sensitive stretch-activated channels in the Merkel cell-neurite complex, which are related to the Na+ influx via voltage-gated Na+ channels and/or the Ca2+ influx through HVA Ca2+ channels.

Mechanoreceptors and length of the patellar ligament after Ho-YAG laser treatment: a long-term follow-up in rabbits.

We studied the shortening and the number of mechanoreceptors in the patellar ligament up to 18 months after Ho-YAG irradiation of the ligament's surface in 35 rabbits. The ligaments shortened an average 13% immediately after irradiation. After treatment, we divided the rabbits into a mobilized or immobilized group. At 2 weeks and 12 months after treatment both groups showed no shortening of the ligament, as compared to the intact ligament while that in the mobilized group had elongated at 12 months. Fewer Pacinian and Ruffini corpuscles were found in the irradiated ligaments than in the intact ones at 2 weeks after treatment, but we found no difference between irradiated and intact ligaments at 18 months after treatment.

Conduction velocity of the spinothalamic tract in humans as assessed by CO(2) laser stimulation of C-fibers.

We measured the conduction velocity (CV) of C-fibers in the spinothalamic tract (STT) following stimulation with a CO(2) laser using a new method. We delivered non-painful laser pulses to tiny areas of the skin overlying the vertebral spinous processes at different levels from the 7(th) cervical (C7) to the 12(th) thoracic (T12), and recorded cerebral evoked potentials in 11 healthy men. We used the term "ultra-late laser evoked potentials" (ultra-late LEPs), since the peak latency was much longer than that for conventional LEPs related to Adelta-fibers following painful laser stimulation (late LEPs). The mean CV of C-fibers in the STT was 2.2+/-0.6 m/s, which was significantly lower than the CV of the Adelta-fibers (10.0+/-4.5 m/s). This technique is novel and simple, and should be useful as a diagnostic tool for assessing the level of spinal cord lesions.

[Study of the photodynamic effect of new photosensitizers on the single neuron]. / Issledovanie fotodinamicheskogo deistviia novykh fotosensibilizatorov na izolirivannuiu nervnuiu kletku.

We studied reactions of isolated crayfish mechanoreceptor neurons to photodynamic effects of various photosensitizers: methylene blue, chlorins e6 and p6, sulfated allumophthalocyanin Photosens, Janus green B, protoporphyrin IX, and two derivatives of hematoporphyrin IX, Photoheme and Photosan-3. The neurons were irradiated by a helium-neon laser (632.8 nm, 0.3 Wt/cm3) after 30-min photosensitization. They proved to be very sensitive to the photodynamic effect: When the cells stained by photosensitizers at nanomolar concentrations were irradiated, their firing activity underwent irreversible changes and they died. The dynamics of the firing activity of the neurons depended on the photosensitizer type and concentration. Photosens, Photoheme and chlorin p6 proved to be the most efficient.

Afferent projections of infrared-sensitive sensilla in the beetle Melanophila acuminata (Coleoptera: Buprestidae).

Beetles of the genus Melanophila are able to detect infrared radiation by using specialized sensilla in their metathoracic pit organs. We describe the afferent projections of the infrared-sensitive neurons in the central nervous system. The axons primarily terminate in the central neuropil of the fused second thoracic ganglia where they establish putative contacts with ascending interneurons. Only a few collaterals appear to be involved in local (uniganglionic) circuits. About half of the neurons send their axons further anterior to the prothoracic ganglion. A subset of these ascend to the subesophageal ganglion, and about 10% project to the brain. Anatomical similarities suggest that the infrared-sensitive neurons are derived from neurons supplying mechanosensory sensilla. The arborization pattern of the infrared afferents suggests that infrared information is processed and integrated upstream from the thoracic ganglia.

Preliminary study of low-level laser for treatment of long-standing sensory aberrations in the inferior alveolar nerve.

PURPOSE: The incidence of inferior alveolar nerve (IAN) damage during removal of third molar teeth has been reported to be as high as 5.5% and up to 100% during sagittal split osteotomy. Sensory aberrations in the IAN persisting for longer than 6 months leave some degree of permanent disability. The purpose of this double-blind, clinical trial was to examine the effects of low-level laser (LLL) treatment using a GaAIAs laser (820 nm, Rønvig, Denmark) on touch and temperature sensory perception after a long-standing postsurgical IAN injury. PATIENTS AND METHODS: Thirteen patients were divided into two groups, one of which received real LLL (4 x 6 J per treatment along the distribution of the IAN to a total of 20 treatments) and the other placebo LLL. The degree of mechanoreceptor injury as assessed by Semmes Weinstein Monofilaments (North Coast Medical, San Jose, CA) were comparable in the two groups before treatment. The degree of thermal sensitivity disability as assessed using a Thermotester (Somedic AB, Stockholm, Sweden) to examine the indifferent temperature threshold was also comparable between the two groups before LLL. RESULTS: Subsequent to LLL, the real laser-treated group showed a significant improvement in mechanoreceptor sensory testing (P = .01) compared with the placebo group, as manifested by a decrease in load threshold (g) necessary to elicit a response from the most damaged area. In addition, the real LLL group reported a subjective improvement in sensory function. There was no significant improvement in thermal sensitivity post-LLL for either the real or placebo laser-treated groups. CONCLUSION: It was concluded that LLL can improve mechanoreceptor perception in long-standing sensory aberrations in the IAN.

[The action of low-intensity infrared laser radiation on skin afferents]. / Deistvie nizkointenzivnogo infrakrasnogo lazernogo izlucheniia na kozhnye afferenty.

Laser radiation modulates functional characteristics of mechanoreceptors. This inhibits impulse activity of the nerve endings from pain focus and reduces pain sensitivity of skin afferents, excitability of conductive nerve fibers. Changes in sensitivity become more pronounced with increasing duration of the exposure. By means of reflectory mechanism laser radiation may cause other general reactions of the body and enhance immune response of the skin.