Optimal pulse length of insonification for Piezo1 activation and intracellular calcium response.

Ultrasound (US) neuromodulation, especially sonogenetics, has been demonstrated with potential applications in noninvasive and targeted treatment of various neurological disorders. Despite the growing interest, the mechanism for US neuromodulation remains elusive, and the optimal condition for eliciting a neural response with minimal adverse effect has not been identified. Here, we investigate the Piezo1 activation and intracellular calcium response elicited by acoustical streaming induced shear stress under various US exposure conditions. We find that Piezo1 activation and resultant intracellular calcium response depend critically on shear stress amplitude and pulse length of the stimulation. Under the same insonification acoustic energy, we further identify an optical pulse length that leads to maximum cell deformation, Piezo1 activation, and calcium response with minimal injury, confirmed by numerical modeling of Piezo1 channel gating dynamics. Our results provide insight into the mechanism of ultrasonic activation of Piezo1 and highlight the importance of optimizing US exposure conditions in sonogenetics applications.

Red and near-infrared light evokes Ca2+ influx, endoplasmic reticulum release and membrane depolarization in neurons and cancer cells.

Low level light therapy uses light of specific wavelengths in red and near-infrared spectral range to treat various pathological conditions. This light is able to modulate biochemical cascade reactions in cells that can have important health implications. In this study, the effect of low intensity light at 650, 808 and 1064 nm on neurons and two types of cancer cells (neuroblastoma and HeLa) is reported, with focus on the photoinduced change of intracellular level of Ca2+ ions and corresponding signaling pathways. The obtained results show that 650 and 808 nm light promotes intracellular Ca2+ elevation regardless of cell type, but with different dynamics due to the specificities of Ca2+ regulation in neurons and cancer cells. Two origins responsible for Ca2+ elevation are determined to be: influx of exogenous Ca2+ ions into cells and Ca2+ release from endoplasmic reticulum. Our investigation of the related cellular processes shows that light-induced membrane depolarization is distinctly involved in the mechanism of Ca2+ influx. Ca2+ release from endoplasmic reticulum activated by reactive oxygen species generation is considered as a possible light-dependent signaling pathway. In contrast to the irradiation with 650 and 808 nm light, no effects are observed under 1064 nm irradiation. We believe that the obtained insights are of high significance and can be useful for the development of drug-free phototherapy.

Long-term transcriptomic and proteomic effects in Sprague Dawley rat thyroid and plasma after internal low dose 131I exposure.

BACKGROUND: Radioiodide (131I) is commonly used to treat thyroid cancer and hyperthyroidis.131I released during nuclear accidents, have resulted in increased incidence of thyroid cancer in children. Therefore, a better understanding of underlying cellular mechanisms behind 131I exposure is of great clinical and radiation protection interest. The aim of this work was to study the long-term dose-related effects of 131I exposure in thyroid tissue and plasma in young rats and identify potential biomarkers. MATERIALS AND METHODS: Male Sprague Dawley rats (5-week-old) were i.v. injected with 0.5, 5.0, 50 or 500 kBq 131I (Dthyroid ca 1-1000 mGy), and killed after nine months at which time the thyroid and blood samples were collected. Gene expression microarray analysis (thyroid samples) and LC-MS/MS analysis (thyroid and plasma samples) were performed to assess differential gene and protein expression profiles in treated and corresponding untreated control samples. Bioinformatics analyses were performed using the DAVID functional annotation tool and Ingenuity Pathway Analysis (IPA). The gene expression microarray data and LC-MS/MS data were validated using qRT-PCR and ELISA, respectively. RESULTS: Nine 131I exposure-related candidate biomarkers (transcripts: Afp and RT1-Bb, and proteins: ARF3, DLD, IKBKB, NONO, RAB6A, RPN2, and SLC25A5) were identified in thyroid tissue. Two dose-related protein candidate biomarkers were identified in thyroid (APRT and LDHA) and two in plasma (DSG4 and TGM3). Candidate biomarkers for thyroid function included the ACADL and SORBS2 (all activities), TPO and TG proteins (low activities). 131I exposure was shown to have a profound effect on metabolism, immune system, apoptosis and cell death. Furthermore, several signalling pathways essential for normal cellular function (actin cytoskeleton signalling, HGF signalling, NRF2-mediated oxidative stress, integrin signalling, calcium signalling) were also significantly regulated. CONCLUSION: Exposure-related and dose-related effects on gene and protein expression generated few expression patterns useful as biomarkers for thyroid function and cancer.

Effect of X-rays on transcript expression of rat brain microvascular endothelial cells: role of calcium signaling in X-ray-induced endothelium damage.

Radiation-induced brain edema is a serious adverse effect of radiotherapy. Although there are many causes of radiation-induced brain edema, the pathogenesis is not clear and clinical treatment is not ideal. Therefore, knowing the differential expression of the brain microvascular endothelial cell (BMEC) transcriptome after brain radiotherapy may shed light on the pathogenesis of radiation-induced brain edema. The present study used RNA-Seq technique to identify 383 BMEC transcripts differentially expressed (many 2-fold or higher; P < 0.05) between control and X-ray-treated primary cultured rat BMECs. Compared with controls, X-ray-treated BMECs had 183 significantly up-regulated transcripts and 200 significantly down-regulated transcripts. The differentially expressed genes were associated with the biological processes of the cell cycle, apoptosis, vascular permeability, and extracellular junctions. The functional changes identified in the X-ray-treated BMECs included Ca2+ signaling, phosphoinositide 3-kinase-Akt signaling, and methionine degradation. These results indicated that transcript expression was substantially affected by radiation exposure and the proteins encoded by these differentially expressed genes may play a significant role in radiotherapy-induced brain edema. Our findings provide additional insight into the molecular mechanisms of radiation-induced brain edema and may be helpful in the development of clinical treatment of this adverse reaction to radiotherapy.

Permanent Photodynamic Activation of the Cholecystokinin 2 Receptor.

The cholecystokinin 2 receptor (CCK2R) is expressed in the central nervous system and peripheral tissues, playing an important role in higher nervous and gastrointestinal functions, pain sensation, and cancer growth. CCK2R is reversibly activated by cholecystokinin or gastrin, but whether it can be activated permanently is not known. In this work, we found that CCK2R expressed ectopically in CHO-K1 cells was permanently activated in the dark by sulfonated aluminum phthalocyanine (SALPC / AlPcS4, 10-1,000 nM), as monitored by Fura-2 fluorescent calcium imaging. Permanent CCK2R activation was also observed with AlPcS2, but not PcS4. CCK2R previously exposed to SALPC (3 and 10 nM) was sensitized by subsequent light irradiation (> 580 nm, 31.5 mW·cm-2). After the genetically encoded protein photosensitizer mini singlet oxygen generator (miniSOG) was fused to the N-terminus of CCK2R and expressed in CHO-K1 cells, light irradiation (450 nm, 85 mW·cm-2) activated in-frame CCK2R (miniSOG-CCK2R), permanently triggering persistent calcium oscillations blocked by the CCK2R antagonist YM 022 (30 nM). From these data, it is concluded that SALPC is a long-lasting CCK2R agonist in the dark, and CCK2R is photogenetically activated permanently with miniSOG as photosensitizer. These properties of SALPC and CCK2R could be used to study CCK2R physiology and possibly for pain and cancer therapies.

Visible light excited ratiometric-GECIs for long-term in-cellulo monitoring of calcium signals.

Genetically Encoded Calcium Indicators (GECIs) are powerful molecular tools for monitoring calcium (Ca2+) signaling in the cytosol and organellar compartments. However, currently available ratiometric indicators that allow measurements of resting Ca2+ levels have limitations in long-term Ca2+ imaging. They either are ultraviolet (UV)-excited ones with strong photo-toxicity, or have poor performance. To overcome this hurdle, we developed a set of visible light excited ratiometric-GECIs (VR-GECIs) based on existing mono-colored GECIs. With performance comparable to their corresponding mono-color prototypes, this set of VR-GECIs enables long-term measurements of intra-cellular or intra-organellar Ca2+ signals. Using these VR-GECIs together with a newly developed off-line analysis tool, we achieved long-term measurements of Ca2+ homeostasis of moving or dividing cells. Our tools may find broad applications in decoding Ca2+-modulated physiological or pathological processes.

Characterization of C2C12 cells in simulated microgravity: Possible use for myoblast regeneration.

Muscle loss is a major problem for many in lifetime. Muscle and bone degeneration has also been observed in individuals exposed to microgravity and in unloading conditions. C2C12 myoblst cells are able to form myotubes, and myofibers and these cells have been employed for muscle regeneration purposes and in myogenic regeneration and transplantation studies. We exposed C2C12 cells in an random position machine to simulate microgravity and study the energy and the biochemical challenges associated with this treatment. Simulated microgravity exposed C2C12 cells maintain positive proliferation indices and delay the differentiation process for several days. On the other hand this treatment significantly alters many of the biochemical and the metabolic characteristics of the cell cultures including calcium homeostasis. Recent data have shown that these perturbations are due to the inhibition of the ryanodine receptors on the membranes of intracellular calcium stores. We were able to reverse this perturbations treating cells with thapsigargin which prevents the segregation of intracellular calcium ions in the mitochondria and in the sarco/endoplasmic reticula. Calcium homeostasis appear a key target of microgravity exposure. In conclusion, in this study we reported some of the effects induced by the exposure of C2C12 cell cultures to simulated microgravity. The promising information obtained is of fundamental importance in the hope to employ this protocol in the field of regenerative medicine.

Response Latency Tuning by Retinal Circuits Modulates Signal Efficiency.

In the visual system, retinal ganglion cells (RGCs) of various subtypes encode preprocessed photoreceptor signals into a spike output which is then transmitted towards the brain through parallel feature pathways. Spike timing determines how each feature signal contributes to the output of downstream neurons in visual brain centers, thereby influencing efficiency in visual perception. In this study, we demonstrate a marked population-wide variability in RGC response latency that is independent of trial-to-trial variability and recording approach. RGC response latencies to simple visual stimuli vary considerably in a heterogenous cell population but remain reliable when RGCs of a single subtype are compared. This subtype specificity, however, vanishes when the retinal circuitry is bypassed via direct RGC electrical stimulation. This suggests that latency is primarily determined by the signaling speed through retinal pathways that provide subtype specific inputs to RGCs. In addition, response latency is significantly altered when GABA inhibition or gap junction signaling is disturbed, which further supports the key role of retinal microcircuits in latency tuning. Finally, modulation of stimulus parameters affects individual RGC response delays considerably. Based on these findings, we hypothesize that retinal microcircuits fine-tune RGC response latency, which in turn determines the context-dependent weighing of each signal and its contribution to visual perception.

Astrocyte Viability and Functionality in Spatially Confined Microcavitation Zone.

Blast-induced traumatic brain injury (bTBI) can result in cell/tissue damage and lead to clinical and neuropsychiatric symptoms. Shock waves from a blast propagate through the brain and initiate cascades of mechanical and physiological events that can adversely affect the brain function. Although studies using animal models and brain slices have shown macroscale changes in the brain tissue in response to blast, systematic elucidation of coupling mechanisms is currently lacking. One mechanism that has been postulated and demonstrated repeatedly is the blast-induced generation and subsequent collapse of micron-size bubbles (i.e., microcavitation). Using a custom-designed exposure system, we have previously reported that upon collapsing of microbubbles, astrocytes exhibited changes in the cell viability, cellular biomechanics, production of reactive oxygen species, and activation of apoptotic signaling pathways. In this paper, we have applied microfabrication techniques and seeded astrocytes in a spatially controlled manner to determine the extent of cell damage from the site of the collapse of microbubbles. Such a novel experimental design is proven to facilitate our effort to examine the altered cell viability and functionality by monitoring the transient calcium spiking activity in real-time. We now report that the effect of microcavitation depends on the distance from which cells are seeded, and the cell functionality assessed by calcium dynamics is significantly diminished in the cells located within ∼800 µm of the collapsing microbubbles. Both calcium influx across the cell membrane via N-type calcium channels and intracellular calcium store are altered in response to microcavitation. Finally, the FDA-approved poloxamer 188 (P188) was used to reconstitute the compromised cell membrane and restore the cell's reparative capability. This finding may lead to a feasible treatment for partially mitigating the tissue damage associated with bTBI.

Photobiomodulation Affects Key Cellular Pathways of all Life-Forms: Considerations on Old and New Laser Light Targets and the Calcium Issue.

After 50 years of studies on photobiomodulation (PBM), there is still so much to investigate to understand the laser light-nonplant cells interactions. The current scientific knowledge allows to say that the phenomena induced by PBM are based on cellular pathways that are the key points of cell life. The mitochondria chromophores, also present on the bacterial membrane, the calcium channels, ion that regulates the life-and-death cellular processes, as well as the TRP family, whose genes have been found in protozoa and suggest that its basic mechanism evolved long before the appearance of animals, seem to be elective targets in photobiomodulatory events by wavelengths from 600 up to 980 nm. The ambiguous resulting cellular communication way, mediated by ATP, ROS and/or calcium, leads to cell manipulation, which modifies its metabolism and whose response connects all life-forms from bacteria to vertebrates. Because of the Giano-Bifronte features of ROS and calcium, as well as the fine balance of energetic mitochondrial processes, whose alteration is responsible for several diseases, the PBM can show unpredictable results and it requires scrupulous approach to avoid cellular damages. However, when carefully applied, PBM is able to improve nonhealthy cell's responses and represents a reliable support in human and veterinary medicine.

The Essential Role of Ca2+ Signals in UVB-Induced IL-1ß Secretion in Keratinocytes.

UVB-induced skin damage is attributable to reactive oxygen species, which are triggered by intracellular Ca2+ signals. However, exactly how the reactive oxygen species are triggered by intracellular Ca2+ upon UVB irradiation remains obscure. Here, we show that UVB induces Ca2+ signals via sequential generation of the following Ca2+ messengers: inositol 1,4,5-trisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose. UVB induced H2O2 production through NADPH oxidase 4 activation, which is downstream to inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate. H2O2 derived from NADPH oxidase 4 activated CD38 to produce cyclic ADP-ribose. UVB first evoked the pannexin channel to release ATP, which acts on P2X7 receptor to generate inositol 1,4,5-trisphosphate. Inhibitors of these messengers, as well as antioxidants, blocked UVB-induced Ca2+ signals and IL-1ß secretion in keratinocytes. Furthermore, ablation of CD38 and NADPH oxidase 4 protected against UVB-induced inflammation and IL-1ß secretion in the murine epidermis. These results show that UVB induces IL-1ß secretion through cross-talk between Ca2+ and reactive oxygen species, providing insight towards potential targets against UVB-induced inflammation.

Variations of Intracellular Ca2+ Mobilization Initiated by Nanosecond and Microsecond Electrical Pulses in HeLa Cells.

GOAL: Herein, the variations in transient Ca2+ mobilizations in HeLa cells exposed to a single, non-thermal pulsed electric field (PEF) are described. METHODS: Three PEF waveforms categorized by pulse duration and intensity were used to deduce the kinetics involved in Ca2+ mobilization. A fast microscopic fluorescent imaging system and a fluorescent molecular probe were used to observe transient intracellular Ca2+ mobilization after pulse exposure. The sources and pathways in the transient Ca2+ mobilizations were investigated using an inhibitor of inositol-1,4,5-trisphosphate receptor (IP3R) on the endoplasmic reticulum (ER) along with a Ca2+-free buffer. RESULTS: When exposed to the 10-µs-long PEF, the Ca2+ concentration increased mainly at the cathodic region near the membrane. However, Ca2+ concentration increased at both anodic and cathodic regions when Na+ concentration in the buffer was reduced. Ca2+ concentration increased only in the presence of extracellular Ca2+. CONCLUSION: These results suggest that the 10-µs PEF takes a large amount of extracellular Na+ into the cell through the electropermeabilized plasma membrane, especially at the anodic side, resulting in the suppression of the Ca2+ influx. On the contrary, the 20-ns-long PEF increased Ca2+ concentration in the surrounding region of the nucleus only in the presence of extracellular Ca2+. The PEF exposure with inhibition of the IP3R indicates that increased Ca2+ ions are released from the ER via the activated IP3R. SIGNIFICANCE: These mechanisms could induce specific cell responses, such as Ca2+ oscillations, Ca2+ waves, and Ca2+ puffs.

Astrocytes integrate and drive action potential firing in inhibitory subnetworks.

Many brain functions depend on the ability of neural networks to temporally integrate transient inputs to produce sustained discharges. This can occur through cell-autonomous mechanisms in individual neurons and through reverberating activity in recurrently connected neural networks. We report a third mechanism involving temporal integration of neural activity by a network of astrocytes. Previously, we showed that some types of interneurons can generate long-lasting trains of action potentials (barrage firing) following repeated depolarizing stimuli. Here we show that calcium signaling in an astrocytic network correlates with barrage firing; that active depolarization of astrocyte networks by chemical or optogenetic stimulation enhances; and that chelating internal calcium, inhibiting release from internal stores, or inhibiting GABA transporters or metabotropic glutamate receptors inhibits barrage firing. Thus, networks of astrocytes influence the spatiotemporal dynamics of neural networks by directly integrating neural activity and driving barrages of action potentials in some populations of inhibitory interneurons.

Calcium, a pivotal player in photodynamic therapy?

Photodynamic therapy combines three non-toxic components: light, oxygen and a photosensitizer to generate singlet oxygen and/or other ROS molecules in order to target destruction of cancer cells. The damage induced in the targeted cells can furthermore propagate to non-exposed bystander cells thereby exacerbating the damage. Ca2+ signaling is strongly intertwined with ROS signaling and both play crucial roles in cell death. In this review we aimed to review current knowledge on the role of Ca2+ and ROS signaling, their effect on cell-cell propagation via connexin-linked mechanisms and the outcome in terms of cell death. In general, photodynamic therapy results in an increased cytosolic Ca2+ concentration originating from Ca2+ entry or Ca2+ release from internal stores. While photodynamic therapy can certainly induce cell death, the outcome depends on the cell type and the photosensitizer used. Connexin channels propagating the Ca2+ signal, and presumably regenerating ROS at distance, may play a role in spreading the effect to neighboring non-exposed bystander cells. Given the various cell types and photosensitizers used, there is currently no unified signaling scheme to explain the role of Ca2+ and connexins in the responses following photodynamic therapy. This article is part of a Special Issue entitled: Calcium signaling in health, disease and therapy edited by Geert Bultynck and Jan Parys.

Kinesin light chain-4 depletion induces apoptosis of radioresistant cancer cells by mitochondrial dysfunction via calcium ion influx.

Kinesins act as molecular microtubule-dependent motor proteins and have various important cellular functions related to cell division, intracellular transport, and membrane trafficking. However, the function of kinesin light chain 4 (KLC4) in cancer, especially radioresistance, has not been previously described. Thus, we investigated KLC4 function in lung cancer cells and radioresistant R-H460 cells by analyzing alterations in radiosensitivity after gene knockdown with siRNA and by evaluating cellular phenotypes and xenograft tumor growth. KLC4 was upregulated in human lung cancer cell lines. Moreover, in paired clinical specimens of lung cancer patients, KLC4 expression was significantly higher in tumor tissues than in paired adjacent normal tissues. Fluorescence-activated cell sorting (FACS) analysis showed that apoptosis rates and cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase-3 levels in KLC4-knockdown lung cancer cells were significantly increased compared with those in control cells. Colony formation decreased as the radiation dose increased in KLC4-knockdown lung cancer cells, demonstrating an essential role for KLC4 in radioresistance. Importantly, KLC4 silencing suppressed tumor growth in an in vivo xenograft model, accompanied by increased apoptosis. Finally, KLC4-knockdown cells exhibited impaired mitochondrial respiration, increased mitochondrial reactive oxygen species production, and enhanced mitochondrial calcium uptake, resulting in mitochondrial dysfunction. Thus, KLC4 as a kinesin superfamily-targeted therapy may represent a novel, effective anticancer strategy, particularly for patients showing radioresistance.

A study of wound repair in Dictyostelium cells by using novel laserporation.

We examined the mechanism of cell membrane repair in Dictyostelium cells by using a novel laser-based cell poration method. The dynamics of wound pores opening and closing were characterized by live imaging of fluorescent cell membrane proteins, influx of fluorescent dye, and Ca2+ imaging. The wound closed within 2-4 sec, depending on the wound size. Cells could tolerate a wound size of less than 2.0 µm. In the absence of Ca2+ in the external medium, the wound pore did not close and cells ruptured. The release of Ca2+ from intracellular stores also contributed to the elevation of cytoplasmic Ca2+ but not to wound repair. Annexin C1 immediately accumulated at the wound site depending on the external Ca2+ concentration, and annexin C1 knockout cells had a defect in wound repair, but it was not essential. Dictyostelium cells were able to respond to multiple repeated wounds with the same time courses, in contrast to previous reports showing that the first wound accelerates the second wound repair in fibroblasts.

Reactive Oxygen Species Produced by a Photodynamic Effect Induced Calcium Signal in Neurons and Astrocytes.

Photodynamic therapy (PDT) leads to production of reactive oxygen species (ROS) and cell destruction due to oxidative stress. We used photodynamic effect of photosensitizer radachlorin to unravel the effect of photo-induced oxidative stress on the calcium signal and lipid peroxidation in primary culture of cortical neurons and astrocytes using live cell imaging. We have found that irradiation in presence of 200 nM of radachlorin induces calcium signal in primary neurons and astrocytes. Photo-induced neuronal calcium signal depends on internal calcium stores as it was still observed in calcium-free medium and could be blocked by depletion of endoplasmic reticulum (ER) stores with inhibitor of sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) thapsigargin. Both inhibitors of phospholipase C activity U73122 and water-soluble analogue of vitamin E Trolox suppressed calcium response activated by PDT. We have also observed that the photodynamic effect of radachlorin induces lipid peroxidation in neurons and astrocytes. This data demonstrate that lipid peroxidation induced by PDT in neurons and astrocytes leads to activation of phospholipase C that results in production of inositol 1,4,5-trisphosphate (IP3).

Curcumin regulates intracellular calcium release and inhibits oxidative stress parameters, VEGF, and caspase-3/-9 levels in human retinal pigment epithelium cells.

In this study, we aimed to observe whether curcumin (cur), a polyphenolic compound derived from the dietary spice turmeric, a yellow substance obtained from the root of the plant Curcuma longa Linn, has any protective effect against blue light irradiation in human retinal pigment epithelium (ARPE-19) cells. For this purpose, we evaluated the intracellular calcium release mechanism, poly ADP ribose polymerase (PARP), procaspase-3/-9 protein expression levels, caspase activation, and reactive oxygen species levels. ARPE-19 cells were divided into four main groups, such as control, cur, blue light, and cur + blue light. Results were evaluated by Kruskal-Wallis and Mann-Whitney U tests as post hoc tests. The cells in cur and cur + blue light samples were incubated with 20 µM cur. Blue light exposure was performed for 24 h in an incubator. Lipid peroxidation and cytosolic-free Ca2+ [Ca2+]i concentrations were higher in the blue light exposure samples than in the control samples; however, their levels were determined as significantly lower in the cur and cur + blue light exposure samples than in the blue light samples alone. PARP and procaspase-3 levels were significantly higher in blue light samples. Cur administration significantly decreased PARP and procaspase-3 expression levels. Reduced glutathione and glutathione peroxidase values were lower in the blue light exposure samples, although they were higher in the cur and cur + blue light exposure samples. Caspase-3 and -9 activities were lower in the cur samples than in the blue light samples. Moreover, vascular endothelial growth factor (VEGF) levels were significantly higher in the blue light exposure samples. In conclusion, cur strongly induced regulatory effects on oxidative stress, intracellular Ca2+ levels, VEGF levels, PARP expression levels, and caspase-3 and -9 values in an experimental oxidative stress model in ARPE-19 cells.

Extracorporeal Shock Wave Rebuilt Subchondral Bone In Vivo and Activated Wnt5a/Ca2+ Signaling In Vitro.

BACKGROUND: This study aimed to identify the optimal extracorporeal shock wave (ESW) intensity and to investigate its effect on subchondral bone rebuilt in vivo and Wnt5a/Ca2+ signaling in vitro using an osteoarthritis (OA) rat model and bone marrow mesenchymal stem cells (BMMSCs), respectively. METHODS: OA rats treated with (OA + ESW group) or without (OA group) ESW (n = 12/group) were compared with healthy controls (control group, n = 12). Gait patterns and subchondral trabecular bone changes were measured. Western blot and quantitative real-time polymerase chain reaction detected protein expression and gene transcription, respectively. RESULTS: The gait disturbances of OA + ESW group were significantly improved compared with the OA group at 6th and 8th weeks. The micro-CT analysis indicated that the BMD, BSV/BV, BV/TV, Tr.S, and Tr.Th are significantly different between OA group and OA + ESW group. Expression of Wnt5a was increased rapidly after ESW treatment at 0.6 bar and peaked after 30 min. CONCLUSIONS: ESW were positive for bone remodeling in joint tibial condyle subchondral bone of OA rat. ESW prevented histological changes in OA and prevented gait disturbance associated with OA progression. Optimal intensity of ESW induced changes in BMMSCs via activation of the Wnt5a/Ca2+ signaling pathway.

Optogenetic approach for targeted activation of global calcium transients in differentiated C2C12 myotubes.

Excitation-contraction coupling in muscle cells is initiated by a restricted membrane depolarization delimited within the neuromuscular junction. This targeted depolarization triggers an action potential that propagates and induces a global cellular calcium response and a consequent contraction. To date, numerous studies have investigated this excitation-calcium response coupling by using different techniques to depolarize muscle cells. However, none of these techniques mimic the temporal and spatial resolution of membrane depolarization observed in the neuromuscular junction. By using optogenetics in C2C12 muscle cells, we developed a technique to study the calcium response following membrane depolarization induced by photostimulations of membrane surface similar or narrower than the neuromuscular junction area. These stimulations coupled to confocal calcium imaging generate a global cellular calcium response that is the consequence of a membrane depolarization propagation. In this context, this technique provides an interesting, contactless and relatively easy way of investigation of calcium increase/release as well as calcium decrease/re-uptake triggered by a propagated membrane depolarization.