laser photocoagulation is widely used for treating various retinal diseases , including diabetic retinopathy , branch retinal vein occlusion , and central serous retinopathy . unfortunately , conventional laser treatments are destructive procedures used to create whole - layer damage to retinal tissue . such invasive treatments are not suitable for macular diseases , including macular edema , because laser scars generally grow larger and create permanent scotomas . laser scar enlargement , subretinal fibrosis , and subretinal neovascular membranes have been reported as serious complications of laser treatment [ 48 ] . micropulse lasers were invented to reduce laser - induced burns that can occur with conventional laser treatment . lasers in the micropulse mode exert pulses of short laser applications , thereby diminishing the heat generated at the target site . in fact , retinal coagulation scars are not visible upon fundoscopic examination , autofluorescent imaging , or optical coherence tomography [ 11 , 12 ] when the eyes are treated with a subthreshold micropulse laser . many studies have shown the clinical efficacy of this treatment [ 10 , 1317 ] ; however , few studies have investigated the molecular mechanisms responsible for fluid absorption from retinal tissue . although thermal stimulation of the retinal pigment epithelium ( rpe ) is thought to be the key trigger for fluid absorption from an edematous retina , the molecular mechanisms of this process are not yet understood . notably , lavinsky et al . found slight morphological changes following laser irradiation with a low - energy output in dutch - belted rabbits . these changes were reversible ( i.e. , they did not cause permanent damage ) when photocoagulation spots ( visible burns ) were barely visible on the retinal surface . these data suggested that biological cascades within the rpe may contribute to the therapeutic effects of sublethal photocoagulation during the treatment of macular edema . heat shock protein ( hsp ) family members , which act as chaperone proteins , aid in refolding denatured proteins and are protective against apoptosis and inflammation [ 2024 ] . in particular , elevated expression of hsp is known to occur with increases in temperature . upregulation of hsp70 expression induced by laser irradiation is thought to play an important role in the improvement of macular edema . intriguingly , nondamaging laser irradiation induces hsp70 expression within the rpe of hemizygous c57bl/6 mice , whereas low - energy laser irradiation induces rpe cell death in dutch - belted rabbits . unfortunately , whether sublethal laser energy induces hsp70 expression in human retinal tissue is still unknown . in this study , as a first step towards understanding the molecular mechanisms through which micropulse laser application affects retinal tissue , we constructed a laser irradiation system to directly expose cultured human retinal pigment epithelial arpe-19 cell layers to a sublethal dose of laser energy . the expression of hsp70 at both the mrna and protein levels was examined over time . the arpe-19 immortal human rpe cell line   at passage 19 ( atcc crl-2303 , lot number : 60279299 ) was purchased from american type culture collection ( manassas , va , usa ) , and all experiments were performed using the cells at passages 22 through 26 . cells were maintained in dulbecco 's modified eagle 's medium : nutrient mixture f-12 ( dmem / f-12 , gibco , life technologies , carlsbad , ca , usa ) supplemented with 10% fetal bovine serum ( one shot fbs ; gibco , life technologies ) and 1% penicillin - streptomycin ( pen strep ; gibco , life technologies ) at 37c in a 5% co2 incubator . cell culture media were replaced 2 - 3 times a week . for laser applications , 3  10 cells were seeded on the glass bottoms ( 12  mm in diameter ) of 35  cm glass - bottomed culture dishes ( asahi techno glass , tokyo , japan ) and left to stand for 30  min . two milliliters of dmem / f-12 was then added , and media were replaced on the fourth day of culture . diode lasers are primarily used for targeting of rpe cells in photocoagulation treatment in the clinical setting . therefore , we constructed an experimental system that made it possible to irradiate a fully confluent layer of cultured cells . in order to directly irradiate cultured cell layers with a laser , as is performed in the clinical setting , a slit - lamp adapter for an 810  nm diode laser ( oculight sl ; iris medical , mountain view , ca , usa ) was placed on top of a slit - lamp system created by cutting a slit - lamp microscope barrel ( 900 bm ; haag - streit diagnostics , bern , switzerland ) and vertically fixing it onto a culture dish . after laser irradiation , cells were inspected with a confocal microscope ( tcs sp8 ; leica microsystems , mannheim , germany ) equipped with a life cell imaging chamber ( stage top incubator ; tokai hit , shizuoka , japan ) and/or a phase - contrast microscope ( dmi3000b ; leica microsystems ) . measurements of photocoagulated areas were performed using an image analyzer ( las 4.2 ; leica microsystems ) and image j 1.47 public domain software ( national institutes of health , bethesda , md , usa ) . a live / dead cell imaging kit ( molecular probe , life technologies ) was used to visualize live and dead cells after laser irradiation . the live / dead reagent , composed of calcein - am and ethidium homodimer-1 , was directly added to conditioned media on cultured cells after laser irradiation and incubated for 15  min at 25c . to discriminate between live and dead cells , the rpe cell layer was inspected with a confocal microscope with the appropriate filters in place . live cells were able to hydrolyze calcein - am and were therefore stained green , while dead cells were permeable to ethidium homodimer-1 and were therefore stained red . all rna isolation experiments were performed using a purelink rna mini kit with dnase ( ambion , life technologies ) according to the manufacturer 's instructions . first - strand cdna was synthesized from 200  ng of total rna using a high capacity rna - to - cdna kit ( applied biosystems , life technologies ) . for quantification of hsp70 mrna , one - twentieth of the above - mentioned cdna was analyzed using taqman fast advanced master mix ( applied biosystems , life technologies ) and taqman gene expression assay probe - primer sets for hspa1a ( hs00359163_s1 ; applied biosystems , life technologies ) . the level of hsp70 mrna was determined by the 2 method   using gapdh ( hs02758991_g1 , applied biosystems , life technologies ) as an internal control . cells were fixed with 4% paraformaldehyde ( thermo fischer scientific , waltham , ma , usa ) and permeabilized using 0.1% triton x-100 . cells were treated with image - it fx signal enhancer ( molecular probe , life technologies ) , blocked in 10% normal goat serum ( molecular probe , life technologies ) , and incubated with an anti - hsp70 antibody ( 3a3 ; abcam , cambridge , uk ) or anti - gapdh antibody ( loading control ; abcam ) . these primary antibodies were visualized with alexa fluor 488 goat anti - mouse igg antibody ( molecular probe , life technologies ) , which served as a secondary antibody . samples were counterstained with slowfade gold antifade mountant with 4,6-diamidino-2-phenylindole ( dapi ; molecular probe , life technologies ) . student 's t - tests and mann - whitney u tests were used to test mean differences for statistical significance . each experiment was conducted more than three times . differences with p values of less than 0.05 cells were irradiated on glass - bottomed dishes via a dichroic mirror and a perpendicular laser ( figures 1(a ) and 1(b ) ) . in order to uniformly expose the cell layer to laser energy , several methods of spreading black paper under culture dishes when performing laser irradiation in arpe-19 cells have been previously reported [ 29 , 30 ] . additionally , culture media without phenol red were used to avoid blocking of the emitted diode laser light . the culture dish was placed on a mechanical stage so that the dish could be moved into the proper laser irradiation position in a flat x - y plane . the stage was moved in 0.1  mm steps , and the cell layer was accurately irradiated with the laser in equal intervals . the position of the laser irradiation site was indicated by a red light pointing in the direction of the x- or y - axis . the position along the z - axis was then determined using the focal point of the laser . the focal point was defined as the position in which the laser aiming beam was most clearly projected onto the surface of the black paper . when a cell layer was irradiated with a conventional continuous - wave mode laser , the laser irradiation site and its periphery gradually changed into a coagulation - like form . the formation of sparse annular intercellular spaces around the laser irradiation site was also observed in bright - field images obtained with a confocal microscope 2  h after laser irradiation ( figure 1(c ) and supplementary material 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/729792 ) . these cell groups in the bright - field images remained viable , as indicated by green fluorescence staining with calcein - am , an indicator of cell survival . in contrast , the laser irradiation site and nearby cells were dead , as indicated by red fluorescence staining with ethidium homodimer-1 , an indicator of cell death ( figure 1(d ) ) . a laser provides intermittent irradiation in the micropulse mode , with the percentage of irradiation time set at a fixed number . the duty cycle is 15% , with the laser irradiating for 0.3  ms during each 2.0  ms period . when the total micropulse laser exposure duration was 1000  ms , the duty cycle was repeated 500 times ( figure 2(a ) ) . figure 2(b ) shows phase - contrast images of irradiation sites 2  h after micropulse laser irradiation was performed with the following laser settings : duty cycle of 15% , laser exposure duration between 500 and 1000  ms , and laser power between 600 and 800  mw . laser irradiation was performed at 1  mm intervals along the x- and y - axes on each cell layer . laser exposure duration was shortened with each 1  mm position change along the x - axis , but laser power remained constant . laser power was decreased with each 1  mm position change along the y - axis , but laser exposure duration remained constant . when the laser power was fixed at 800  mw , the coagulation area gradually decreased as the laser exposure time was shortened ( 1000 to 500  ms : 184  8.8 to 81  13.5  m ) . however , coagulation - like lesions were no longer visible with phase - contrast microscopy when the laser power was 600  mw and the laser exposure duration was 600  ms . additionally , coagulation area proportionally increased with laser power within the range examined ( spearman 's correlation coefficient : r > 0.8 , p < 0.05 , figure 2(c ) ) . based on the observed changes in micropulse laser irradiation lesions in a cultured cell layer ( figure 2(b ) ) , the optimal , reproducible sublethal laser settings were determined to be a laser exposure duration of 1000  ms , a duty cycle of 15% , and a laser power of 600  mw . bright - field images , captured with a confocal microscope , revealed that cell morphology changed in sublethal photothermal stimulation sites where intercellular spaces were sparse ( figure 2(d ) ) . however , almost all cells within sublethal photothermal stimulation sites were alive , even 24  h after micropulse laser irradiation ( figure 2(d ) ) . additionally , the coagulation - like area was significantly smaller for lesions created in the micropulse mode than for those created in the continuous - wave mode under the same energy conditions ( mann - whitney u test : p < 0.001 , figures 2(e ) and 2(f ) ) . to investigate whether sublethal photocoagulation induced hsp expression , which is important in reducing macular edema , we used qrt - pcr to examine the expression levels of hspa1a mrna , which encodes hsp70 protein , in a cultured cell layer irradiated with a sublethal irradiation dose using a micropulse laser ( figure 2(d ) ) . cells were examined until 24  h after irradiation from each of the 81 irradiation positions in the x - y plane . in the pretreated irradiation group , the expression of hsp70 mrna was only slightly increased over baseline , even 24  h after irradiation ( figure 3(a ) ) . because the clinical efficacy of macular edema treatment has been shown to be better with high - density micropulse laser irradiation than with standard laser treatment , we also examined the effects of varying the number of laser irradiation lesions on hspa1a mrna levels by changing the spacing of laser irradiation lesions on the cultured cell layer , which resulted in an increase in the number of lesions from 81 to 477 . as expected , a significant increase in hspa1a mrna expression was observed 3  h after irradiation in cell layers with a higher number of lesions ( figure 3(b ) ) . actual hsp70 protein levels in cultured cell layers were also examined after sublethal laser irradiation using immunocytochemistry with an anti - hsp70 antibody ( figure 3(c ) ) . immunofluorescent signals were present in concentric areas around laser irradiation sites . however , gapdh , used as an endogenous control for qrt - pcr analyses of hspa1a mrna , was uniformly present throughout the cultured cell layer . the presence of cells within laser irradiation sites was confirmed with dapi counterstaining . even 24  h after photothermal stimulation , hsp70 immunostaining persisted at laser irradiation sites . in this study , we established a system in which one layer of densely cultured cells could be irradiated with a diode laser . moreover , we confirmed that sublethal micropulse photothermal stimulation induced hsp70 expression in a cultured layer of arpe-19 cells . previous reports in the literature have shown that the size of coagulation lesions created using a continuous - wave laser is dependent on laser duration and power in dutch - belted rabbits [ 31 , 32 ] . our in vitro study results are consistent with this and revealed that this is also true for coagulation - like lesions made with a micropulse laser ( figures 2(b ) and 2(c ) and supplementary material 2 ) . using the same amount of energy , more tissue damage occurred in lesions created with a continuous - wave laser than in those created with a micropulse laser ( figures 2(e ) and 2(f ) ) . it is thought that micropulse lasers cause less damage than continuous - wave lasers because the pulses of very short , intermittent energy may allow for heat dissipation within tissue . although numerous clinical studies have shown the efficacy of subthreshold micropulse laser treatments , the laser settings ( i.e. , laser power and pulse duration ) used in these studies have not been consistent [ 10 , 1317 ] . moreover , the optimal laser energy for subthreshold laser treatments has not yet been established , and surgeons often use continuous - wave laser modes for titrating laser settings [ 16 , 17 , 33 ] . our study revealed that laser thermal damage caused by continuous - wave and micropulse laser modes differed ( figures 2(e ) and 2(f ) ) . our results also suggested that laser power and exposure time titrations for the treatment of retinal disease should be performed using subthreshold micropulse photocoagulation and not continuous - wave laser photocoagulation . sublethal micropulse photothermal stimulation sites had cellular groups with sparse intercellular spaces that did not have any dead cells , even 24  h after laser irradiation , as determined using cell viability assays ( figure 2(d ) ) . we presumed that these intercellular spaces represented the loss of cell - to - cell adhesion in the rpe layer , which may contribute to a dysfunctional blood retinal barrier in vivo , directly increasing fluid and molecule movement between the retina and choroidal tissues . restoring the blood retinal barrier with photothermal stimulation may lead to improvements in macular edema . future research on proteins related to rpe cell adhesion and rpe tight junctions ( e.g. , occludin , claudin , and zonula occludens-1 ) is needed in rpe cell layers . several studies have shown that heated tissue remaining viable after conventional continuous - wave laser treatment undergoes a stress response , which includes production of beneficial intracellular biological factors ( e.g. , pedf , tsp-1 , sdf1 , and hsp ) with antiangiogenic and restorative functions [ 25 , 30 , 34 ] . because hsp may block the activity of apoptotic and inflammatory pathways that cause cellular damage [ 2024 ] , upregulation of hsp70 expression induced by laser irradiation is thought to be important for reducing macular edema following clinical laser therapy [ 25 , 26 ] . in the current study , sublethal micropulse photothermal stimulation was shown to induce upregulation of hsp70 , the major hsp in mammalian cells . moreover , expression of hsp70 mrna , quantified by real - time qrt - pcr , was temporarily upregulated in preconditioned cultured cell layers for 12  h following laser irradiation . immunostaining indicated that expression of hsp70 proteins in cultured cell layers occurred concentrically around laser - irradiated sites . this study is the first to report hsp70 expression and upregulation in cultured cells following sublethal micropulse laser energy application . the beneficial effects of subthreshold micropulse photocoagulation for diabetic macular edema are not observed immediately in the clinical setting but begin to become evident 3 months following therapy [ 10 , 1317 ] . therefore , sublethal micropulse photothermal stimulation that induces hsp70 upregulation may facilitate rpe remodeling and contribute to improvements in macular edema . first , we examined a monolayer of cultured cells , which may not have the same vertical heat conduction properties as an intact eye . secondly , arpe-19 cells exhibit some characteristics and features that are different from those of human rpe cells . therefore , the laser irradiation system developed and used in this study should be further examined in arpe-19 cells and in human rpe cells derived from retinal tissue and induced pluripotent stem cells . in conclusion , while the therapeutic mechanism underlying the efficacy of micropulse laser therapy has not yet been determined , our data showed that hsp70 may be a trigger for improving rpe health and function . moreover , hsp70 may be used as a biological marker to determine therapeutic laser levels for subthreshold micropulse photocoagulation . in addition , testing laser irradiation systems in a cultured layer of human rpe cells is advantageous because changes in cell morphology , molecular composition , cellular transcriptomes , and cellular proteomes after sublethal photothermal stimulation can be precisely assessed using live imaging microscopy , dna microarrays , and mass spectrometry techniques .