low - level - laser therapy ( lllt ) is an increasingly used and effective complementary treatment in clinic , especially for wound healing , anti - inflammation , and pain relief . mast cells ( mcs ) , the vital immune cells , in dermis paly necessary roles in the process of wound healing . mcs have recently been identified to have important anti - inflammatory functions in vivo to some extent , despite their well - known role as promoter of inflammation . subcutaneous or mucosal mcs in vivo have been suggested to be involved in anti - inflammatory   and wound healing [ 47 ] reactions of lllt . recently , subcutaneous mcs have been shown to participate in the initial process of pain relief by laser acupuncture , an increasingly and frequently applied version of lllt . in medicine , red and near - infrared ( nir ) lasers , with wavelengths between 600 and 1,000  nm , are frequently applied in lllt because laser light at these wavelengths can penetrate tissues in the millimeter range as absorption by human skin is low . the approximate skin transmission depth of red light is about 1.52  mm , mainly reaching mcs residing in cutaneous and subcutaneous skin layers . mcs in skin may , therefore , participate in lllt mechanisms by being directly activated during red - laser irradiation . this hypothesis has been supported by some investigations in vitro : activation of degranulation [ 12 , 13 ] , [ ca]i elevation [ 1214 ] , as well as triggering of whole - cell membrane currents   by red - laser irradiation were demonstrated in mcs . extracellular nucleotides are important players in regulating inflammatory responses through binding to purinergic p2 receptors present on all inflammatory cells , including mcs . activation of p1 receptors by adenosine or other agonists can promote the wound healing process . extracellular atp is not only the endogenous agonist for several p2 receptors but also the precursor of other related nucleotides . the effect of irradiation on extracellular atp content of mcs is unknown . in the periphery , mcs morphologically   and functionally interact with nerve endings , in which purinergic signaling has a major role , and a crosstalk between mcs and peripheral neurons via purinergic signals exists . whether this crosstalk could be modulated by low - level irradiation , we assessed the responses of extracellular atp content of mcs and drg neurons to low - level red - laser irradiation and further explored the underlying mechanisms . the aim of this study is to better understand of the role of atp purinergic signaling in lllt effects at the cellular level . j. h. butterfield ( mayo clinic , rochester , mn , usa ) and cultured as described previously . in brief , the cells were incubated in phenol - red - free imdm medium , supplemented with 2  mm l - glutamine , 25  mm hepes , 10% ( v / v ) fetal bovine serum 1% penicillin , and streptomycin , in a 95% humidity - controlled incubator ( model : 310 , thermo , thermo electron , waltham , usa ) with 5% co2 at 37c . briefly , adult sprague - dawley rats ( 280320  g ) were sacrificed by co2 asphyxiation . 30  ml ca / mg free hank 's balanced salt solution ( hbss ) was injected into peritoneal cavity of each rat . after vigorous agitation of the abdominal area for 2  min , injected hbss was collected and centrifuged at 400  g for 5  min with swing - out rotors . ammonium - chloride - potassium lysing buffer ( 150  mm nh4cl , 10  mm khco3 , and 0.1  mm edta - na2 ) was used to break erythrocytes . the cell pellet was well resuspended with 8  ml 70% isotonic percoll and gently overlaid with 2  ml rpmi 1640 medium . mcs stayed at the bottom fraction after centrifugation of the cell suspension at 580  g for 15  min . the viability and the purity were identified with 0.4% trypan blue and 0.5% toluidine blue ( tb ) . the cell density was adjusted as needed ( see section 2.6 ) and cells were equilibrated in the incubator for 2  h. isolation of rat drg neurons was modified according to burkey et al . briefly , adult sprague - dawley rats ( 150170  g ) were sacrificed by co2 asphyxiation . hibateral drgs were dissected from thoracic to lumbar regions of the vertebral column and placed in oxygenated ca / mg free hbss . the connective tissue sheath around the ganglia was removed and then incubated in 3  ml of f12 medium containing 1.25  mg / ml collagenase ia , 300  u deoxyribonuclease iv ( dnase ) , and 0.05% trypsin ix - s at 37c for 50  min . at the end of the incubation period , 8  ml f12 medium containing 10% horse serum was added to terminate the enzymatic reaction . ganglia pellet was collected by centrifugation ( 200  g , 2  min ) , resuspended in f12 medium , and mechanically agitated through a fire - polished glass pasteur pipet until the suspension of dissociated cells was homogeneous . nerve fragments were discarded by centrifugation at 60  g for 2  min , and isolated individual cells of the bottom pellet were resuspended in f12 medium once more and transferred to a 35  mm petri dish to culture for 2  h. at the end of the incubation , after nonneuronal cells were attached to the bottom , the unattached sensory neurons were collected for equilibration in the incubator for 2  h. 4-week - old male sprague - dawley rats ( shanghai experimental animal center of chinese academy of sciences , shanghai , china ) weighing 120150  g were deeply anesthetized with ether . each animal was placed in an airtight container with several cotton balls soaked with ether ( sinopharm chemical reagent co. , ltd . , hair in the area of the lateral tibia was shaved , and the skin was cut open with scissors to expose connective tissues , which were acutely separated with blunt forceps and scissors before incubation in bath solution ( bs ) ( see section 2.3 ) for 1  h. tissue slices comprised three connective layers , with one loose layer sandwiched between two dense layers . the loose connective tissue was exposed by stretching the two dense layers in opposite direction . thereafter , the tissue slices were fixed by tiny needles to hot glue support on the chamber bottom . to identify mcs , fixed slices were incubated in 0.5% tb for 10  min and subsequently washed 3 times with 95% ethanol . the present study was performed in accordance with the guidelines of the animal care and use committee of shanghai university of traditional chinese medicine ( certificate no . the bs for tissue slices contained ( in mm ) 137 nacl , 2.7 kcl , 2 cacl2 , 5 mgcl2 , 5.6 glucose , and 10 hepes , ph 7.4 ( adjusted with naoh ) . the bs for hmc-1 cells was comprised of the following ( in mm ) : 150 nacl , 5 kcl , 2 cacl2 , 5 mgcl2 , 4d - sorbitol , and 10 hepes , ph 7.4 ( adjusted with naoh ) . osmolality of the solutions was 310  mosm / kg , as verified with osmometer ( model : 3300 , micro osmometer , advanced instruments inc . , culture mediums , hbss , serum , antibiotic , calcium green-1 am , and pluronic f-127 were bought from invitrogen company ( usa ) ; all other chemicals were purchased from sigma company ( usa ) . reagents were prepared in stock solutions and kept at 20c and diluted in bs or culture medium before use . in some atp - accessing experiments , hmc-1 cells were pretreated with nem or bapta or arl 67156 for 20  min before luminescence measurements . according to the kubelka - munk theory , red - light irradiance decreases by > 90% at a depth of 1.52  mm ( see also [ 10 , 25 ] ) . hence , in our present experiments , we started with a dose of 17  j / cm , 5% of the dose level used in our previous animal studies . the laser device and irradiation parameters used for hmc-1 cells and tissue slice are presented in table 1 . for rat peritoneal mcs and neurons , the laser power density was adjusted to 18  mw / cm , and then 20  min exposure generated 21  j / cm . in atp - related experiments , equilibrated cell suspensions in 1.5  ml eppendorf tubes ( see section 2.6 ) were exposed to laser light entering the tube vertically , in order to expose the entire suspension to irradiation . in light and fluorescence imaging experiments , light was introduced at 60-degree angle to tissue slices or hmc-1 cells , and all cells viewed under microscope were irradiated . to monitor possible heating effects of laser irradiation , temperature changes of perfusion bs induced by red - laser amounted to only 0.2c during 15  min irradiation . in order to exclude the possibility that reagents were modified by the laser light , the absorption spectrums of 100  m nem , 25  m bapta , and 100  m arl 67156 were scanned . none of the drugs showed significant absorbance at the 657  nm laser light . to investigate the effects of laser irradiation on rat skin mc degranulation , tissue slices were fixed in a perfusion chamber and micrographs were captured under upright light microscope ( model : nf1 , nikon , japan ) . to estimate the [ ca]i of hmc-1 cells , calcium green-1 fluorescence was measured as described previously . briefly , hmc-1 cells were grown on glass cover - slips coated with poly - l - lysine before mounting in the perfusion chamber . 5  mm calcium green-1 am stock solution was dissolved in 20% ( w / v ) pluronic f-127 . hmc-1 cells were loaded in phenol - red - free imdm containing 4  m calcium green-1 am for 45  min ; then the loaded cells were superfused with bs . micrographs were captured under inverted light microscope ( model : te2000-u , nikon , japan ) by ccd video camera ( orca - er , hamamatsu photonics , hamamatsu , shizuoka , japan ) . images were digitized and averaged ( 3 frames ) , background corrected , and analyzed by an image - processing system ( wasabi software , hamamatsu photonics , hamamatsu , shizuoka , japan ) . fluorescence intensities of individual cells in the viewing field were ascertained by averaging the image intensities collected from regions of interest within each cell . graphs were colored by image j software ( national institutes of health , bethesda , md , usa ) . all microscopy images were taken with 40x objective magnification ( cfi super plan fluor elwd , 0.6 in n.a . ) . atp content was quantified by bioluminescence assay measuring light output from luciferin - luciferase reactions . to evaluate extracellular atp content , 100  l aliquots of dispersed cell suspensions were placed in 1.5  ml eppendorf tubes for hmc-1 cells or into 96-well plate for isolated cells . after equilibration for 2  h in the incubator , all samples were divided into nonirradiated control and irradiated groups exposed to laser light for different irradiation time periods . to measure atp level in the samples , 100  l luciferin - luciferase assay mix that was diluted with dilution buffer and was adjusted to isotonicity with mannitol was added to each sample , and light emission was measured immediately by luminometer ( glomax 20/20 , promega , madison , wisconsin , usa , for hmc-1 cells or synergy mx , biotek , winooski , usa for isolated cells ) . to determinate the intracellular atp content , 50  l aliquots of dispersed cell suspensions were transferred into 1.5  ml eppendorf tubes . 50  l phenol - red - free imdm and 100  l somatic cell atp - releasing reagents were added to each aliquoted 50  l sample . after briskly swirling , 100  l mixtures were transferred to each 1.5  ml eppendorf tube containing 100  l luciferin - luciferase assay and light emission was measured immediately by luminometer , as above . luciferin - luciferase luminescence was calibrated versus atp standards before and after all sample measurements . the data were analyzed by origin software ( originlab , northampton , ma , usa ) and expressed as averages  sem . the n values give the number of measurements obtained from different samples of cells ; the n values present the number of independent experiments . differences between sample averages were compared by one - way anova , and p < 0.05 was considered to represent statistically significant difference . in control hmc-1 cell suspension samples of 3.5  10  cells / ml the extracellular atp content was 0.41  0.06  fmoles / cell ( n = 27 ) . relative extracellular atp increase induced by 3  min irradiation was 26.8  7.2% of control ( p < 0.01 , n = 8 ; figure 1 ) , and it was blocked by 100  m nem , an inhibitor of exocytosis ( see table 2 ) . this indicates that the elevated extracellular atp originated from a cell regulated release process and not from cell death . it is interesting that the response tended to decline with longer irradiation time of 5  min ( with an increase of only 10.3  3.3% compared to control , n = 9 ) . this phenomenon has been described as the biphasic dose response to low - level light irradiation discovered in 1985 . it was confirmed in numerous subsequent reports that biostimulation can be obtained at weaker doses , while stronger doses result in bioinhibition . extracellular atp content depends on three metabolic steps : synthesis , release , and hydrolysis . in various cultured cell types , red and nir - light irradiations have been reported to promote atp synthesis [ 29 , 30 ] that is indexed by intracellular atp content . intracellular atp in nonirradiated control hmc-1 cells amounted to 14.2  1.8  fmoles / cell ( n = 30 ) . it increased slightly during the first minute of irradiation and was significantly enhanced , by 45.5  22.6% compared to control values , after 3  min exposure ( p < 0.05 , n = 15 ; figure 2 ) . similar to the extracellular atp responses , 5  min irradiation induced a smaller increase of somatic atp content ( by 18.0  13.7% compared to control , n = 9 ) . the coherent response of intracellular with extracellular atp content may suggest that atp synthesis partially contributes to elevated external atp content during irradiation . we found that laser irradiation significantly elevated [ ca]i in hmc-1 cells ( n = 1517 cells , p < 0.01 ) . an example experiment is shown in figure 3(a ) , which illustrates the increase in ca fluorescence at different irradiation times . increase of relative fluorescence intensity became apparent after 1  min irradiation and amounted to 3.5  0.6% ( n = 17 cells , p < 0.01 ) of the controls . it rose to 18.0  1.0% ( n = 15 cells , p < 0.01 ) and 21.5  1.1% ( n = 15 cells , p < 0.01 ) with irradiation times of 3 and 5  min , respectively ( figure 3(b ) ) . in order to judge whether irradiation - induced [ ca]i elevation correlates with the atp release , intracellular free ca in hmc-1 cells the results showed that pretreatment of hmc-1 cells with 25  m bapta blocked the extracellular atp rise induced by 3  min irradiation ( table 2 ) . this suggests that the extracellular atp level depends on [ ca]i - dependent atp release . in order to confirm the effects of red - laser irradiation on wild - type mcs connective tissue slices isolated from the lateral side of the rat tibia contained mcs at high density .   figure 4(a ) shows two mcs in a slice section ; resting cells exhibited smooth and complete membranes . once the slices were exposed to red - laser irradiation for 5  min , some mcs in these slices started to degranulate , displaying rough membrane and releasing granules that scattered around the cells ( figure 4(b ) ) . since peritoneal mcs are of the connective tissue phenotype as skin mcs and the available number of isolated peritoneal mcs is much higher than skin mcs , rat peritoneal mcs were taken to determinate the effects of red - laser irradiation on atp mobilization . similar to the responses observed with hmc-1 cells , 21  j / cm irradiation of rat peritoneal mcs also showed tendency of enhanced extracellular atp content , although the increase did not reach statistical significance ( figure 5 ) . for nonirradiated neurons , extracellular atp content was 0.8  0.1  fmoles / cell ( n = 28 ) . in contrast to mcs , extracellular atp level was attenuated by 26.0  2.6% in drg neurons at 21  j / cm irradiation ( n = 27 , p < 0.01 , figure 6 ) . to eliminate the possibility that atp outside neuronal cells could be degraded by the laser irradiation , the results showed that atp was not affected by laser irradiation ( table 2 ) . considering that multiple ecto - atpases are expressed on drg neurons , we wondered whether the suppressive effect resulted from modulation of ecto - atpase activity by irradiation . figure 6 illustrates the response of extracellular atp content of drg neurons to irradiation in the presence of the nonspecific ecto - atpases inhibitor arl 67156 . 100  m arl 67156 partially attenuated the irradiation - induced inhibition of extracellular atp ( n = 15 , p < 0.05 , figure 6 ) . this suggests that at least to some extent the irradiation reduced external atp level of drg neurons by stimulating the ecto - atpases activity . the mechanisms of the remaining suppressive effects in the presence of arl 67156 need further exploration . in vivo mcs have been demonstrated to play a role in lllt for anti - inflammatory , wound healing , as well as pain relief . not only the total number of mcs but also degranulation ratios were increased by red - laser irradiation . in in vivo tests , increasing degranulation [ 12 , 13 ] , histamine release [ 34 , 35 ] , and [ ca]i [ 1214 , 34 , 35 ] as well as whole - cell membrane currents   were demonstrated in mcs irradiated by green- , blue- , or red - laser . extracelluar atp is an important autocrine / paracrine mediator to modulate system functions in various tissues by binding to p2 receptors . red - laser irradiation - induced elevation of extracellular atp level of mcs has been illustrated in our previous work . in the present study , we confirmed such potentiated effects and demonstrated dose dependency . extracellular atp content is influenced by several processes , including synthesis , release , and hydrolysis . in our study , the irradiation - induced coherent response of intracellular and extracellular atp content suggests that the stimulating effects of irradiation on extracellular atp can partially be attributed to enhance atp synthesis . promoting atp synthesis by red and nir - light irradiation has been reported in various cultured cell types [ 29 , 30 ] . the absorption of monochromatic light by components of the cellular respiratory chain , such as cytochrome c oxidase , has been reported [ 35 , 36 ] . besides providing more atp to be loaded into the secretory vesicles , increased atp synthesis also will facilitate atp - driven transporters such as ca and na / k atpase . whether the respiratory chain is involved in the processes described in our work the idea of  photo - switched  ligands that modulate the membrane channels gating has been put forward [ 37 , 38 ] . our results also revealed that irradiation could elevate [ ca]i in mcs , which is required as the trigger signal for atp release , leading to increased extracellular atp level . our experiments with bapta - loaded cells provided further evidence that the extracellular atp level depends on the [ ca]i rise . since extracellular atp is the endogenous agonist of p2x receptors which allow extracellular ca to enter the cells , and mcs themselves express p2x receptors , we hypothesize that the atp secreted from mcs could produce positive feedback by autocrine activation of the p2 receptors . such a mechanism could amplify irradiation effects in local areas to treat local disorders , for example , to act in anti - inflammatory reactions   in wound - healing , or in local pain relief . like the subcutaneous mcs , peripheral neuritis in skin is also in a depth that can be reached by irradiation during lllt . in our work , irradiation attenuated extracellular atp content of drg neurons , which is opposite to the response of mcs . the depressive effects of irradiation on peripheral nerve system have been reviewed . since soluble and membrane - attached ecto - atpases , that hydrolyze extracellular atp , exist in peripheral nervous system , including drg peripheral endings , modulation of ecto - atpases of drg neurons by irradiation could explain the effect on extracellular atp levels . in our work , 100  m arl 67156 abolished the irradiation - induced depression extracellular atp of drg neurons suggesting an upregulation of ecto - atpases on drg neurons . the ecto - atpases hydrolyze extracellular atp into amp , the precursor of adenosine which is a vital mediator involved in pain relief by binding to a1 receptors [ 42 , 43 ] . the peripheral nervous system is the main domain of purinergic receptors , including a1 receptors . thus modulation of ecto - atpases by irradiation might be one of the mechanisms underlying analgesic effects of lllt . there exists crosstalk via purinergic signals between them . concerning the opposite responses to irradiation in mcs and neurons , we hypothesize that stimulated ecto - atpase on drg neurons could hydrolyze part of the extracellular atp released by the mcs during lllt . low - level red - laser irradiation of mcs and drg neurons has opposite effects on extracellular atp content . irradiation of mcs enhanced extracellular atp level in a dose - dependent manner , which is brought about by increased intracellular atp synthesis and [ ca]i . for drg neurons , irradiation depressed the extracellular atp content due to upregulation of ecto - atpases . the opposite responses of these two cell types indicate complex mechanisms underlying lllt that need further investigations .
low - level - laser therapy ( lllt ) is an effective complementary treatment , especially for anti - inflammation and wound healing in which dermis or mucus mast cells ( mcs ) are involved . in periphery , mcs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes . 
 whether extracellular atp , an important purine in purinergic signaling , of mcs and neurons could be modulated by irradiation remains unknown . in this study , 
 effects of red - laser irradiation on extracellular atp content of mcs and dorsal root ganglia ( drg ) neurons were investigated and underlying mechanisms were explored in vitro . 
 our results show that irradiation led to elevation of extracellular atp level in the human mast cell line hmc-1 in a dose - dependent manner , which was accompanied by elevation of intracellular atp content , an indicator for atp synthesis , together with [ ca2+]i elevation , a trigger signal for exocytotic atp release . in contrast to mcs , irradiation attenuated the extracellular atp content of neurons , which could be abolished by arl 67156 , a nonspecific ecto - atpases inhibitor . 
 our results suggest that irradiation potentiates extracellular atp of mcs by promoting atp synthesis and release and attenuates extracellular atp of neurons by upregulating ecto - atpase activity . 
 the opposite responses of these two cell types indicate complex mechanisms underlying lllt .