European Journal of Histochemistry 2018; volume 62:2867

The Dendrobaena National Science Foundation statement. veneta (Annelida): Through their genetic and evolutionary Correspondence: Prof. Stefano Benedicenti, peculiarity, comparable to those of scientif- Department of Surgical Sciences and A new experimental-organism ically accredited models, D. veneta allows Integrated Diagnostic, University of Genoa, for photobiomodulation the effect of laser therapies by multidiscipli- Largo R. Benzi 10, 16132 Genova, Italy. and wound healing nary methods, at various degree of com- E-mail: [email protected] plexity and costs to be investigated. Key words: Low Level Laser Therapy; laser 1,2 2 irradiation; scarring; anti-inflammatory; tissue Andrea Amaroli, Sara Ferrando, repair; Heat Shock Protein; cell proliferation. Marina Pozzolini,3 Lorenzo Gallus,2 Steven Parker,1 Stefano Benedicenti1 Introduction Contributions: AA, SF, SB, experiments con- cept and design; AA, LG, MP, SF, experiments 1Department of Surgical Sciences and A few years after the first working laser performing, data collection, data analyses; SB, Integrated Diagnostic, University of was invented, Endre Mester and co-work- 1 SP, analysis tools providing; AA, MP, SB, SF, Genoa ers, while applying low-level laser energy SP, manuscript drafting. 2 irradiation to the backs of shaved mice, Department of Earth, Environmental noticed that the shaved hair grew back more and Life Sciences, Laboratory of New Conflict of interest: The authors state no con- quickly on the treated group than the flict of interest and have received no payment Model Organism (NeMoLAB), untreated group. This manipulation of cellu- in preparation of this manuscript. University of Genoa lar behavior using low intensity light 3 Department of Earth, Environmental sources (non-ablative) was referred to as Acknowledgements: We are grateful to Dr. and Life Sciences, Laboratory of Marine photobiomodulation (PBM). Conceptually, Marino Rottigni and Dr. Giulia Damiano for invaluable experimental support. Biotechnology, University of Genoa, PBM and the application of photonic energy Italy of specific individual wavelengths works on Received for publication: 3 November 2017. the principle of inducing a biological Accepted for publication: 15 January 2018. response through energy delivery and trans- only fer into the tissue, modulating biological This work is licensed under a Creative Abstract processes within that tissue and within the Commons Attribution-NonCommercial 4.0 biological system of which that tissue is a International License (CC BY-NC 4.0). Photobiomodulation (PBM) is a manip- part.2 Photonic energy incorporateduse within a ulation of cellular behavior using non-abla- molecular structure, may increase kinetic ©Copyright A. Amaroli et al., 2018 tive low intensity light sources. This manip- energy, activate or deactivate enzymes or Licensee PAGEPress, Italy ulation triggers a cascade of metabolic European Journal of Histochemistry 2018; 62:2867 alter physical or chemical properties of doi:10.4081/ejh.2018.2867 effects and physiological changes resulting main macromolecules. Growth factor in improved tissue repair, of benefit in the response within cells and tissue as a result treatment of tissue injury, degenerative or of increased cellular energy and protein power density or irradiance of the applied autoimmune diseases. PBM has witnessed synthesis, together with a change in cell electromagnetic energy.4,8 In addition, the an exponential increase in both clinical membrane permeability to Ca2+ uptake, may application of PBM using “standard” (hith- instrument technology and applications. It lead to cell proliferation.3-5 This may form erto conventional) hand-piece is determined is therefore of benefit to find reliable exper- part of a cascade of metabolic effects and by its very features. Through beam diver- imental models to test the burgeoning laser physiological changes resulting in gence, the applied energy is distributed over technology for medical applications. In our improved tissue repair associated with an increasing area as the tip-to-tissue dis- work, we proposed the earthworm injuries, degenerative diseases or autoim- tance increases, dramatically affecting ener- Dendrobaena veneta for the study of non- mune diseases.6 However, a lot of confusion gy density at cellular level.8 Laser energy ablative laser-light effects on wound heal- still reigns in this field since to date, the cor- emitted from a standard hand-piece is not ing. In our preliminary work, D. veneta has rect laser parameters and resultant applied evenly distributed across its emitting sur- been shown to be positively affected by energy density that effectively promote cell face. In almost all instances it has a PBM. New tests using D. venetaNon-commercial were set up rescue without significant side effects, are Gaussian profile, so the density of the pho- to evaluate the effectiveness of a chosen still elusive. Limited evidence suggests that tons is significantly higher at the center of 808 nm-64 J/cm2–1W-CW laser therapy the energy can be applied with the same the beam and lower at its outskirts.8,9 In using the AB2799 hand-piece with flat-top efficiency from cellular to organismic order to overcome these problems, PBM bean profile, on the wound healing process level.7 Indeed, if the photonic energy can has witnessed an exponential increase in of the earthworm. Effective outcome was have a photobiomodulatory positive effect both clinical instrument technology and assimilated through examining the macro- on in vitro cells and tissues, that energy applications. It is therefore of benefit to find scopic, histological, and molecular changes must reach target cell at the appropriate reliable experimental models to test the bur- on the irradiated posterior-segment of intensity to be effective in vivo. It is possi- geoning laser technology for medical appli- excised- with respect to con- ble that this may be due to incomplete cations. Recently, a novel hand-piece trols. Three successive treatments, one knowledge surrounding the biochemical (AB2799) with flat-top bean profile has every 24 hours, were concluded as suffi- mechanisms underlying the PBM positive been produced (LAMBDA SpA, Vicenza, cient to promote the wound healing, by effect. Additionally, there is the complex Italy). This particular probe provides homo- effects on muscular and blood vessel con- influence of choice among a large number geneous irradiation and energy density, its traction, decrement of bacteria load, reduc- of operating parameters such as wavelength beam is non-divergent and provides the tion of inflammatory processes and tissue of the applied electromagnetic energy, the same power density over any tip-to-tissue degeneration. D. veneta was demonstrated absorbing chromophores within the target distance from contact to 100 cm (see prod- to be a reliable experimental organism that tissue, the energy density or fluence of the uct characteristics on http://www.lambdas- meets well the 3Rs principles and the applied electromagnetic energy and the pa.com/en/). In our previous papers,10,11 we

[European Journal of Histochemistry 2018; 62:2867] [page 51] Original Paper

proposed the unicellular eukaryotic organ- When mature, they have a visible set of be completed in 100-150 days and 65 days ism Paramecium primaurelia as a model to swollen segments, the clitellum, specialized is the average time to reach sexual test novel PBM therapies and laser tech- for . The anterior part of the maturity.29 nologies. Through these investigations with body, from the mouth to the clitellum (in D. D. veneta can be easily bred in laborato- P. primaurelia, we showed that the higher- veneta roughly 30 segments), contains sev- ry, in standard conditions, without addition fluence (64 J/cm2) using higher-power (1 eral different organs (e.g., ovaries, testes, of manure, by organic soil and veg- W), continuous wave (CW) irradiation, multicellular glands, cerebral ganglia, etable or oatmeal as food.24-29 with the AB2799, is able to induce PBM, nephridia, specialized regions of the ali- affecting mitochondrial activity,9 increasing mentary canal such as pharynx, crop, and Experimental setting oxygen consumption,10 and ATP gizzard). Posteriorly to the clitellum, there D. veneta were obtained from Redbug production,12,13 as well as modulating Ca2+ are several tenths of segments that are S.r.l. (Milan, Italy), acclimated in laboratory fluxes, Ca2+ stored release, nitric oxide pro- anatomically more homogeneous, exclud- into organic soil at a temperature of 22±1°C duction and cilia beating.12-14 In addition, ing the few, very caudal ones.23 In our pre- and oatmeal fed.24 The experimental setting the higher-fluence higher-power irradiation liminary work,24 D. veneta showed to be was performed as summarized in Table 1. by flat-top hand-piece of gametes and promising model, which was positively Adult clitelled selected specimens with a embryos of the sea urchin Paracentrotus affected by PBM. In the present work, we similar length (~150 segments), weight lividus, does not affect the DNA and does focalized our attention to the wound healing (~1.5 g) and color were rinsed with water not generate aberration.15 In the current in the posterior part of D. veneta, where all and anesthetized on an ice block for a few work, we moved from the unicellular to the the segments share the following anatomy. minutes.29 Under sterile laminar flow hood, multicellular level and we propose an ani- The body wall is a muscular structure cov- 30 posterior segments were excised from mal experimental organism to test the pho- ered externally by a cuticle and epidermis the and non-irradiated (control) or tobiomodulatory effect of PBM on wound and internally by the peritoneum.25 The irradiated with a 808 nm diode (Doctor healing. An attempt to find more ethical and anatomical structures in the coelomic cavi- Smile, LAMBDA Spa) using a hand-piece more human closed model organism, which ty, running along the body length, are the with flat-top bean profile (AB2799, Doctor meets well the 3Rs (Replacement, gut, enveloped in a chloragogen layer, the Smile,only LAMBDA Spa), with a fluence of 64 Reduction, Refinement) principles,16 has major dorsal and ventral blood vessels, and J/cm2 and a power of 1 W in CW. Laser irra- emerged through recent decades. The words the ventral nerve cord. Furthermore, a cou- diation was performed with the hand-piece “model organism”, “model ” or ple of nephridia are present laterally in each at 1 mm away from the samples, immedi- 23 “model animal” are more and more used in segment. . In the fluid that fills the coelom- ately after the excisions (0 h) and 24 h and different science fields. Some of these mod- ic cavity are present free coelomocytes;use48 h thereafter. The irradiated and non-irra- els have consequentially been promoted for they are a heterogeneous population of diated excised posterior segments were “human health”, while others have been cells. The main cell types are amebocytes, placed in individual sterile Petri dishes (Ø ignored but without real motivation.17,18 The deriving from the peritoneum, and eleo- 19 cm), with 60 gr of organic soil and 50 accredited models for the study of inflam- cytes, deriving from the chloragogen mL of water. The samples were kept under 26 matory process, wound healing and regen- layer. The body-wall muscle is formed by this experimental condition for up to eration are both Vertebrates (Mus musculus, two layers of helical muscle fibers; they are twelve-days and 20 mL of water was added Rattus norvegicus, Danio rerio, Xenopus elongated mononucleated cells, with spiral- every three days. laevis)19,20 and Invertebrates ly-arranged sarcomeres and well developed, Simple organisms like the worms show 27,28 (Caenorhabditis elegans, Drosophila tubular, sarcoplasmic reticulum. When a high-ability for wound healing and regen- melanogaster).19 However, the responses to the body of the earthworm is transversally eration. Therefore, in our experiments, to inflammatory stress in the mouse model are sectioned both the gut eversion and the for- point out the laser treatments’ effectiveness, not so easily correlated to that of humans.21 mation of a coelomocytes-formed coelomic we chose to excise the 30-posterior seg- 25 In addition, as reviewed by Katz18 “Thomas plug can be involved in wound closure. . ments because the literature reported this Hunt Morgan, the father of Drosophila To reach our goal, we set up new tests on D. condition induces the death of the 100% genetics, never once referred to Drosophila veneta to evaluate the effectiveness of the excised samples, within 15 days, as a conse- 2 as a model organism in his iconic books. He 808 nm-64 J/cm –1W laser therapy irradiat- quence of the lack of healing.30 cited the advantages of choosing Non-commercial the fly ed with the AB2799 hand-piece, on the The irradiated and non-irradiated sam- Drosophila as an experimental organism, wound healing process of that earthworm, ples were analyzed at different time inter- but he never used the word “model”. After by examining the macroscopic, histological, vals for the evaluation of bacteria load, all, he was not studying a model of the and molecular changes on the irradiated macroscopic changes, histochemical and mechanisms of heredity; he was studying posterior-segment of excised-earthworms immunohistochemical analysis, expression the actual mechanism of heredity. Sydney with respect to controls. of HSP70 and telomerase activity. Brenner did not refer to the worm Caeno - rhabditis elegans as a model organism in Sample temperature analysis his classic work on its genetics”. Basically, The temperature of the excised posteri- in a scientific-world where the future of Materials and Methods or segments was measured in 5 points of the medicine is the “Personalized Medicine”,22 earthworm section, before and after the irra- the concept of “model for human health” is Dendrobaena veneta (Phylum: diation by a Testo 826-T4 infrared ther- actually being questioned.18 Therefore, Annelida, Class: Oligochaeta) mometer with laser marking (Testo Spa, according to Katz18 in our work we pro- D. veneta (also sometimes called Milan, Italy). posed D. veneta as an “experimental organ- hortensis) is a robust ism” for the study of non-ablative laser light earthworm that grows very rapidly and may Bacterial load analysis effects on the wound healing. tolerate much wider moisture ranges than The irradiated and non-irradiated Earthworms, as , are segmented many other species; it prefers temperature excised posterior segments were immedi- worms and belong to the group of . in the range of 15-25°C.29 Its life cycle can ately swabbed, under sterile laminar flow

[page 52] [European Journal of Histochemistry 2018; 62:2867] Original Paper

hood, on either Tryptic Soy Agar (15 gr/L teria loads were detected spectrophotomet- reaction was considered as synonymous agar, 15 gr/L peptone from pancreatic digest rically by a Jenway-6405-UV-Vis Spectro - with the death of the organism. casein, 5 gr/L papaicsoy peptone, 5 gr/L photometer (Jenway, Felsted, Dunmow sodium chloride, pH 7.4) or in LB broth (10 Essex, UK). The bacteria load of the exper- Histochemical analysis gr/L tryptone, 5 gr yeast extract, 10 gr/L iments in LB broth was deduced spec- The 24 h-, 48 h- and 72 h-irradiated and sodium chloride, pH 7.4). The Tryptic Soy trophotometrically by a Jenway-6405-UV- non-irradiated excised posterior segments Agar and the LB broth were kept in the Vis Spectrophotometer, at 3, 6 and 24 h were fixed in 4% paraformaldehyde in a 0.1 incubator (Gallenkamp GmbH, UK) at after the swab. M phosphate buffered solution (PBS, pH 22°C (D. veneta’s breeding temperature), 7.4) at 4°C, Paraplast (Bio-Optica Spa, for 24 h. After 24 h the Tryptic Soy Agar Macroscopic changes analysis Milan, Italy) embedded and cut into 5-mm- was photographed and the images acquired The irradiated and non-irradiated thick sections. Histological observations by the Zeiss stereomicroscope with a excised posterior segments were examined were performed using hematoxylin-eosin Cellpad E (TiEsseLab s.r.l., Milan, Italy). by a Zeiss stereomicroscope, immediately (H&E) and Masson’s trichrome. The Alexa The bacteria load was deduced by the meas- after the irradiation or the non-irradiation 488-conjugated lectin wheat germ agglu- urement with ImageJ 1.33J software (t0) and every 24 h from those. The images tinin (WGA; source: Triticum vulgaris, (National Institute of Health, Bethesda, were acquired with a Cellpad E (TiEsseLab binding specificity: N-acetylglucosamine MD, USA) of the bacterized area. In addi- s.r.l.). The vitality of the segments was eval- and N-acetylneuraminic acid, working dilu- tion, the bacterized areas were carefully col- uated by prodding with a metal tip. The tion: 10 mg/mL – Termofisher Scientific, lected, suspended in LB broth and the bac- stimulation reactions were observed. No Waltham, MA, USA) was used. DNA pres-

Table 1. Description of the irradiation parameters and experimental design. Irradiation parameters Laser Wavelength Hand-piece Electromagneticonly wave Power Diode 808 nm Flat-top Continuous wave 1 W Irradiation time Fluence Irradiance Distance Treatments 60 sec 64 J/cm2 1 W/cm2 1 mm 0 h, 24 h, 48 h from excision Experimental design use Organism Segments number Weight Segment excised Dendrobaena veneta ~ 150 ~ 1.5 gr 30

Non-commercial

[European Journal of Histochemistry 2018; 62:2867] [page 53] Original Paper

ence was identified, when necessary, by for iCycleriQ Real-Time Detection System Statistical analysis 33 4’,6-diamidino-2-phenylindole (DAPI) flu- (Bio-Rad). The statistical analysis was performed orescence staining. Sections were examined Telomerase activity analysis using a one-way ANOVA followed by the on a Leica DMRB light and epifluorescence Tukey-Kramer multi-comparison test microscope; images were acquired with a The telomerase activity is typically cor- (GraphPadInStat 3) to discriminate statisti- Leica CCD camera DFC420C (Leica, related to cellular proliferation during 34 cally significant results. High significance Heerbrugg, Switzerland). wound healing. level: P<0.001 (***), significance level: Therefore, the 24 h-, 48 h-, 72 h- and 96 HSP70 expression analysis P<0.01 (**), significance level: P<0.05 (*), h-irradiated and non-irradiated excised pos- not-significant level: P>0.05. Each experi- Immune responses to heat shock pro- terior segments were frozen in liquid nitro- teins (HSP) develop in virtually all inflam- gen and their proliferative status was detect- matory diseases and evidence on anti- ed by qPCR using the “Quantitative inflammatory effects of HSP70 are shown Telomerase Detection kit” (Allied Biotech, in literature.31 Inc., Benicia, CA, USA), following the Therefore, the amount of HSP70 manufacture’s instruction. The amount of mRNA (MF093577) in excised posterior extracted protein used in each sample was 1 segments of D. veneta 24 h- and 48 h-irra- μg. To correlate the Ct obtained in the dif- diated and non-irradiated was determined ferent samples, a standard curve was pre- by qPCRusing Chromo 4 apparatus (MJ pared with a 1:5 serial dilution of an Research, Waltham, MA, USA). oligonucleotide (TSR) supplied by the kit First total RNA was extracted from and the enzymatic activity was finally expressed as attomoles per microgram pro- frozen liquid nitrogen tissue samples using Figure 1. Effect of the laser irradiation on RNeasy Mini Kit (QIAGEN, Milan, Italy) tein. Specifically, values for 24 h, 48 h, 72 h the temperature of Dendrobaena veneta and RNase-Free DNase Set (QIAGEN) and 96 h-irradiated samples were compared excised side. The temperature was meas- according to manifacturer’s instructions. to 24 h-, 48 h-, 72 h- and 96 h non-irradiated uredonly before (control) and after (irradiated) Then worm cDNA was synthesized by samples ones. the laser irradiation. iScript reverse transcriptase (Bio-RAD, Hercules, CA, USA) in a final volume of 20 μL using 500 ng of purified total RNA. Subsequently, each PCR reaction was per- use formed in 20 μL containing: 1× master mix iQSYBR®Green (Bio-Rad), 0.2 μM of each primer, and 0.8 μL of synthesized cDNA or negative control. All samples were analyzed in triplicate. The reference gene used for the samples normalization was D. veneta 18S ribosomal RNA gene (KJ911964.1) whose primers sequence were (5′CCGTAACTCT- GATGACTCTG) forforward and (5′CTTC- CTTGGATGTGGTAGC 3′) for reverse while the HSP70 primers sequence were (5’ GGACAACAACCTCCTTGGAA 3’) for forward (5’ CGAAGGTTACCTC- GATCTGG 3’) for reverse. All primers were designed using the software Beacon Designer 7.0 (PREMIER Biosoft International, Palo Alto, CA, USA)Non-commercial and obtained from TibMolBiol (Genoa, Italy). The following thermal conditions were used: initial denaturation for 3 min, fol- lowed by 45 cycles with denaturation at 95°C for 15 s, annealing and elongation at 60°C for 30 s. The fluorescence was meas- ured at the end of each elongation step. The next step was a slow heating (1°C/s) of the amplified product from 55°C to 92°C in order to generate a melting temperature curve. Data analyses were obtained using the DNA Engine Opticon® 3 Real-Time Detection System Software program (ver. Figure 2. Bacterial load analysis in non-irradiated (control) and irradiated excised side of 3.03) and, in order to detect the relative Dendrobaena veneta. A= bacterial colonies grown for 24 h on Tryptic Soy Agar. B= image gene expression of HSP70, compared with a analysis of the area of bacterial colonies grown for 24 h on Tryptic Soy Agar. C= spec- calibrator sample (uncut worm), the com- trophotometric analysis of the bacterial colonies grown for 24h on Tryptic Soy Agar. D= 32 spectrophotometric analysis of bacterial colonies grown for 3 h, 6 h and 24 h in LB broth. parative threshold Ct method was used O.D.= optical density. No-star symbol= P>0.05; *= P<0.05; **= P<0.01; ***= P<0.001. with the software GeneExpression Analysis

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ment was carried out by 3 replications and 5 reduced. The body contraction induced the of the body (Figure 4H), while the non-irra- repetitions. internalization of the alimentary tract, diated wound remained open with the ali- which disappeared from the wound site and mentary tract visible (Figure 4G). the major blood vessel was not visible. The Lastly, 144 h (6 days) after the excision coelomic fluid and the blood flow ceased Results (Figure 4B’). Twenty-four hours after the first laser Evaluation of thermic increment treatment, in the irradiated samples the The temperature of the excised posteri- wound seemed to close and the body wall or segments was 22.0°C±0.2 before the irra- contracted (Figure 4D), while the non-irra- diation and 22.5°C±0.4 afterwards (Figure diated samples remained similar to the pre- 1). The difference between the measures did vious day. not constitute statistical significance Forty-eight hours after the excision and (P>0.05, data not shown). 24 h from the second treatment, the irradiat- ed samples wound continued to recover and Evaluation of bacteria load analysis its color appeared uniform with the rest of The analysis of the excised posterior the body (Figure 4 F). Conversely, the ali- segments swabs showed that the bacterized mentary tract of the non-irradiated sample area on the Tryptic Soy Agar was reduced in remained evaginated, although the body the irradiated samples (-41%) compared to wall was more contracted and both the the non-irradiated posterior segments coelomic fluid and blood were absent (Figure 4E). Figure 3. Dendrobaena veneta survival rate (P<0.001) (Figure 2A, B). The spectropho- expressed as a percentage, in non-irradiat- tometric analyses of the bacterized area Seventy-two hours after the excision and 24 h from the third treatment, the ed (control) and irradiated (3 irradiation) emphasizes these differences, quantifying excised site. No-star symbol= P>0.05; *= the bacteria load of the irradiated sample as wound of irradiated samples regained shape P<0.05;only **= P<0.01; ***= P<0.001. 52% less than the non-irradiated sample and pigmentation in similar form to the rest (P<0.001) (Figure 2C). In addition, the spectrophotometric analysis of the LB bac- terized broth, 3 h, 6 h and 24 h after the use swab, showed that the irradiation reduced the LB bacteria load by about 45%, 40% and 62%, respectively (P<0.001) (Figure 2D). Evaluation of survival rate The reaction to the external stimulus showed that, 6 days after the excision, 100% of both irradiated and non-irradiated excised posterior segments were alive (P>0.05) (Figure 3), with the irradiated excised posterior segments appearing more vital and dynamic. 9 days and 12 days after the excision, 100% of the irradiated samples were healthy, while only 75% and 50% respectively, of the non-irradiated samples appeared vital (P<0.001) (Figure 3). Finally, 16 days after the excision, all theNon-commercial non-irra- diated segments succumbed, whereas 100% of the irradiated segments survived (P<0.001) (Figure 3). Evaluation of macroscopic changes Immediately after the 30 posterior seg- ment excision the analysis by stereomi- croscopy revealed the evagination of the alimentary tract and the major blood vessel. Figure 4. Effect of the laser irradiation on the wound healing of Dendrobaena veneta The coelom cavity was open and the excised side. Images acquired by Zeiss stereomicroscope. A=earthworm immediately after the excision (0 h). B=earthworm immediately after the excision (0 h) and B’, the same coelomic fluid emerged as well as the organism immediately after the first irradiation (0 h). C=non-irradiated (control) earth- blood. The body wall was loose and the worm 24 h after the excision and D, earthworm 24 h after the first irradiation. E=non- wound appearance revealed a circular sec- irradiated (control) earthworm 48 h after the excision and F, earthworm 48h after the tion of diameter similar to the uncut body first irradiation and 24 h after the second irradiation. G= non-irradiated earthworm diameter (Figure 4 A,B). After the first laser 72 h after the excision and H, earthworm 72 h after the first irradiation and 48 h and treatment, performed 2 minutes after the 24 h after the second and the third irradiation, respectively. I=non-irradiated earthworm 144 h after the excision and L, earthworm 144 h, 120 h and 96 h after the first, second excision (0 h), the body exhibited a dorsal- and third irradiation, respectively. at= alimentary tract; cf= coelomic fluid. Bars= 2.3 mm. ventral thinning and the wound diameter

[European Journal of Histochemistry 2018; 62:2867] [page 55] Original Paper

and 96 h from the third and last treatment, (Figure 8A); cytoplasm of cells among the 48 h (Figure 8D) and 72 h (Figure 8F). Also, the irradiated posterior excised segments muscle fibers in the body wall (Figure 8 B- a continuity between WGA-labeled cells of gave an appearance similar to the posterior F); granules in the cytoplasm of epidermal peritoneum and WGA-labeled cells in the side of a non-excised earthworm (Figure cells (Figure 8F). muscle tissue was observed in some slides. 4L), whereas the non-irradiated samples The scattered WGA-labeled cells in the In the laser-treated specimens the WGA- showed evidence of gangrene (Figure 4I). peritoneum were always present and did not labeled cells in the muscle tissue were change their aspect and distribution in dif- almost absent after 24 h (Figure 8C), 48 h ferent specimens. Alternatively, the WGA- (Figure 8E) and 72 h (and three laser treat- Histology - general observations labeled cells among the muscle fibers ments) from the dissection (data not Twenty-four hours after the dissection, changed their distribution over time and dif- shown). The WGA-labeled granules in epi- in the control specimens, the body wall ferent treatments. In control specimens, dermal cells were observed only in control muscles were not contracted, the alimentary they became visible 24 h after the dissection specimens and they were most abundant at tract lumen was in contact with the external (Figure 8B) and increased their presence at 72 h after the dissection (Figure 8F). environment and the alimentary tract wall was everted, forming a wide protrusion (Figure 5A). The blood vessels close to the wound were enlarged (Figure 6A). Twenty-four hours after the dissection and the first laser treatment, the body wall muscles were contracted and the alimentary tract wall was non-everted (Figure 5B). The blood vessels close to the wound revealed a reduced lumen compared to the control (Figure 6B). Forty-eight hours after the dissection, in only control specimens, the body wall muscles were partially contracted and formed an easily-visible bulge close to the wound site. The alimentary tract lumen was open to the external environment, and the alimentary use tract wall was still everted (Figure 5C), as well as the blood vessels being still enlarged (Figure 6C). Forty-eight hours after the dissection and the second laser treatments, the edges of the wound appeared near-apposed and the alimentary tract lumen withdrawn into the body cavity (Figure 5D). The blood ves- sels were still reduced in size (Figure 6D). Seventy-two hours after the dissection, in control specimens, the alimentary tract was no longer everted and in some cases it appeared withdrawn into the body cavity (Figure 5E). Seventy-two hours after the dissection and the third laser treatments, the body wall margins had closed and epidermis Non-commercialcovered the wound site (Figure 5F). Histology - body wall muscles The body wall muscles in control spec- imens presented a progressive degenera- tion, from 24 h to 72 h after the dissection (Figure 7 A,C,E); in particular, gaps appeared among the muscle fibers, with a more abundant presence of collagen. The histology of body wall muscles in laser- treated specimens did not show degenera- tion (Figure 7 B,D,F). Figure 5. Histological section of 30 posterior excised segments of Dendrobaena veneta. Histology - WGA labeling Masson’s Trichrome stain (A-D) or Hematoxylin and Eosin stain (E, F). A, C, E= non-irra- The WGA lectin highlights three differ- diated (control) samples, 24 h, 48 h and 72 h from the excision, respectively. B, D, F = irradiated samples, 24 h, 48 h and 72 h from the excision or, on the other hand after 1, ent structures in the histological sections of 2, 3 irradiations, respectively. *=everted alimentary tract wall. Ce=celom; Il= intestinal D. veneta: the cytoplasm of scattered cells lumen; Mu= muscular tissue. Bars, 300 mm. in the peritoneum, which lines the coelom

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Evaluation of telomerase activity intracellular calcium concentration.28 support the observed immediate effects. Amaroli et al.14 proved the effect of the The histological analysis of samples fixed The telomerase activity increased, com- 2 pared to the uncut (P<0.001), after the exci- 808nm diode laser (64 J/cm , 1 W) therapy 24 h after the first irradiation or non-irradi- on P. primaurelia calcium fluxes and ation, highlights the everted intestine of the sion in both the irradiated and the non-irra- 35 diated posterior segments (P>0.05) and it Sharmizaev showed marked spasmolytic controls, which is folded back to expose the remained constant up to 72 h from the exci- properties of laser irradiation, likely due to inside epithelium. The alimentary tract sion (Figure 9). However, at 96 h the level calcium signaling in the adjacent muscle’s eversion and the formation of a coelomic of telomerase activity was maintained in the cells. Therefore, our data suggest an effect plug are the two classical ways to rapidly of the laser on the cellular muscle calcium close the wound, as described in the earth- irradiated sample, while there was a dramat- fluxes, which result in the D. veneta striate worm .25 Amaroli et al.24 ic decrease in the non-irradiated samples musculature contraction. showed that also D. veneta is able to pro- (P<0.001) (Figure 9). In the moments after irradiation, the duce the coelomic plug and this plug is thin- Evaluation of HSP70 expression PBM decreases the bacteria concentration ner in the excised earthworm exposed to on the top of excised posterior segments, as 808 nm diode laser, 64 J/cm2, 1 W therapy, The HSP70 protein expression increased revealed by our microbiological analysis. than in the control, as a demonstrable con- approximately 10 times in the non-irradiated Literature shows that PBM may modify sequence of a decreased inflammatory sample (control), compared to the uncut growth of wound bacteria, which could process. earthworm, whereas imperceptible, but sta- affect wound healing.36 In particular, flu- In our work, in contrast to the control, tistically significant, change was recorded in 2 2 ences from 4.2 J/cm up to 50 J/cm (CW) the irradiated excised tails did not close the the irradiated earthworm (P<0.001) (Figure and laser light wavelengths in the range of wound through alimentary tract eversion, 10). At 48 h from the excision the non-irradi- 380-810 nm can excite endogenous por- but by a vigorous muscular contraction. The ated earthworm exhibited continued increase phyrins and kill the related pigmented-bac- apposition of the irradiated excised tail in HSP70 expression (P<0.001) and the irra- teria.36,37 body wall flaps provides evidence of the diated sample retained a protein expression We do not know the qualitative effect of consequence of this contraction. In addi- similar to the uncut earthworm (P>0.05) our therapy on the earthworm excised pos- tion, by the Masson’s trichrome stain, a new (Figure 10). terior segments bacteria populations, how- tissueonly mass is identified. This mass is colla- ever, the quantitative effect is clear and its gen-free, while the collagen is evident in the role in the inflammatory process should not flaps existing tissue. The differences Discussion be underestimated. The short-termuse effects between irradiated and non-irradiated (con- The ability to respond to injury and to repair tissue is a fundamental property of multicellular organisms. In the moments after an injury occurs, various intracellular and intercellular pathways must be activat- ed and coordinated, if tissue integrity and homeostasis are to be restored.19 If this response is successful and the injury does not result in the demise of the organism, these processes must be shut down in a pre- cise sequence in the ensuing days as recov- ery progresses. Notwithstanding differences between varying organisms, the three clas- sic stages of wound repair, inflammation, new tissue formation and remodeling, are observed in almost all eukaryotic organ- isms.19. In our work, the effect Non-commercialof the treat- ment with 808 nm diode laser, 64 J/cm2, 1 W (CW), on the wound healing of excised posterior segments of D. veneta are classi- fied in three steps: immediate effects (0 h after irradiation), short-term effects (24 h, 48 h and 72 h after irradiation) and long- term effects (72 h over irradiation). Immediately after treatment the irradiat- ed D. veneta showed evident muscular con- traction at the wound site, pointed out by the dorsal-ventral thinning of the body and the internalization of the alimentary tract, which disappeared from the wound site only in irradiated earthworms. Oligochaeta Figure 6. Histological section of Dendrobaena veneta excised side. Masson’s Trichrome show striate transverse and helical muscula- stain, 24 h after excision, in non-irradiated (control) (A) and irradiated, 1 irradiation, (B) ture arrangement,27 which, like the verte- earthworms. Hematoxylin and Eosin staining, 48 h after excision, in non-irradiated (con- trol) (C) and irradiated, 2 irradiations, (D) earthworms. A-C-D, bars, 100 mm. B, bars 20 brate striate musculature, contracts by m. Ce= celom; Mu= muscular tissue; ▲= blood vessel lumen. changes in calcium fluxes and variation of μ

[European Journal of Histochemistry 2018; 62:2867] [page 57] Original Paper

trol) are also evident, for the major blood ous results with the 808 nm diode laser (64 correlating to muscular atrophy. Cells nor- vessels close to the excised side. In fact, J/cm2, 1 W) therapy, which reduced the D. mally present in the muscle tissue could their diameter is smaller in the irradiated veneta inflammation process, as evident in a become aligned to the WGA positive cells than in the non-irradiated histological sam- decrease of the non-cholinergic acetyl- because of the muscle atrophy. However, ple and the contour edge of blood vessel cholinesterase production.24 the labeling of the amoebocytes has not to lumen is more regular in the lasered tails The modifications of the muscle of the be excluded. In fact, the WGA strongly than in the controls. This data concurs with control samples are also highlighted by the labeled the mesenchymal cells lining the the above-mentioned effect of laser irradia- positive labeling of the WGA; absent (24 h) coelom. These cells are able to produce tion on the muscular contraction and further or very scant (48 h) in the irradiated sam- amoebocytes, which in the earthworm as in supports the possible effect of the laser on ples. The interpretation of this staining in mammalian macrophage, migrate up to the calcium fluxes. The endothelial cells of the both the control-posterior-segment and the inflamed site, phagocyte debris (like dead earthworm are properly considered mio- irradiated-posterior-segment of excised- cells), foreign particles and microorgan- endothelial cells, whose contraction ability earthworms is not clear. However, the pro- isms, promoting wound healing.25 The increase with the increment of blood vessel gressive temporal-increment in the fluores- WGA also labeled little structures into the lumen.37 The earthworm blood vessel con- cence in the controls and the near-absence epidermis of the controls, which are too traction relies on the myofilaments, which in the irradiated samples, suggests that small to be classified as vesicles but also contract by variations of intracellular calci- WGA positive cells promotes alterations DNA-free, so not catalogued as bacteria. um concentration.38,39 It is not unreasonable therefore to assume, as with the major blood vessels, that our laser irradiation promotes a contraction, with a mechanism similar to that hypothesized for the muscular tissue. Our histological analysis shows also other evident differences between irradiated and non-irradiated excised tails. Twenty- only four hours after the excision, and ever more evident in the following days the muscular fibers progressively became distinct from each other, with the consequent increase in the connective tissue amongst them. This use change is clear when observing the longitu- dinal cross-sections of circle musculature, just below the epidermis, as well as with the longitudinal musculature. Conversely, the irradiated sample had a compact muscular wall, with thin trabecular connective among the fibers. It is known that muscular atrophy is a phenomenon occurring in the organism with muscle tissue as conse- quence of senescence, inactivity or patho- logical causes40 and in mammalian species a correlation between inflammatory process- es and muscle atrophy is documented.41 The HSP70 is one of the most conserved pro- teins of “animal kingdom” and its involve- ment in the inflammatory process is well documented.31,42 In fact, HSP70 is involvedNon-commercial in the restoration of cell homeostasis, in the immunoregulation and in the phagocytosis of cellular residue, to promote cell prolifer- ation and wound healing.42 Our data show for the first time the expression of HSP70 in D. veneta. In the control samples its expres- sion was time-dependent and highest at 48 h (+1984% respect to the non-excised earth- worm). The irradiated samples showed increase in HSP70-expression at 24 h but the increment was very small (+60% com- pared to the non-excised earthworm) and at 48 h, after the third irradiation the HSP70- levels were similar to that in the non- Figure 7. Histological section of Dendrobaena veneta excised side. Masson’s Trichrome stain. excised earthworm. Therefore, a reduction A,C,E) Non-irradiated (control) samples, 24, 48 and 72 h from the excision, respectively. in the inflammation through PBM without a B,D,F) Irradiated samples, 24, 48 and 72 h from the excision or, on the other hand after 1, 2 and 3 irradiations, respectively. Ep, epidermal tissue; Mu, muscle tissue; MuC, circular muscle thermal-shock is evident. tissue; MuL, longitudinal muscle tissue; Ct, connective tissue. Scale bars: 5 mm. This assertion is supported by our previ-

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They could be evidence of a virus infection, as reported in Drosophila melanogaster, in which it induces the HSP70 increment.43 However, moving beyond these specula- tions, it is clear that this was present only in the epidermis of the control, progressively destined to gangrene, but not in the irradiat- ed-epidermis. If the first step in wound healing is the control of the inflammation processes to restore homeostasis, the new tissue formation by new cell proliferation is the second. The histochemical analysis as little as at 24 h showed new tissue formation in the irradiated excised earthworm, but our evaluation of the telomerase activity point- ed out an interesting cell behavior. The excise earthworm had an increment of telomerase activity, compared to the non- excised earthworm. Up to 72 h, this incre- ment was similar for both irradiated and non-irradiated D. veneta. At 96 h it decreased drastically in the control, while it remained at high level in the irradiated sam- ples. Actually, the high-level telomerase activity observed at 96 h in the irradiated only organisms cannot be a direct effect of the irradiation (the last irradiation was at 48 h). Therefore, if as reported in literature, the telomerase activity is typically correlated to cellular proliferation during the wound use healing of mammalian tissue,34 we can speculate that the inflammatory processes and the tissue degeneration of non-irradiat- ed excised D. veneta deplete the cell energy and its proliferative potential within the fourth-day. Conversely, the laser irradiation would allow a more equilibrated employ- Figure 8. Histological section of Dendrobaena veneta excised side. The Alexa 488-conju- ment of cell energy, by the mitigating effect gated lectin wheat germ agglutinin (WGA) binds (green fluorescence, white arrows) the N-acetylglucosamine and N-acetylneraminic acids. The DNA is point out by DAPI fluo- on both inflammation and tissue degenera- rescence staining (blue fluorescence). A) The WGA is positive in the cytoplasm of scat- tion and a likely effect on the energetic cel- tered cells in the peritoneum of both non-irradiated and irradiated samples. B,D,F) Non- lular metabolism, in according to our previ- irradiated (control) samples 24, 48 and 72 h after the excision. C,F) Irradiated samples, ous works.9-14 24 and 48 h from the excision or, on the other hand after 1 and 2 irradiations, respective- A foreseeable consequence therefore ly. Ce,celom; S, septum; Ep, epidermal tissue; MuC, circular muscle tissue; MuL, longi- emerges when all data is considered; the tudinal muscle tissue. Scale bars: 50 mm. control specimens become infected, become gangrenous and die, while the irra- diated samples complete the woundNon-commercial healing and tissue remodeling and after twelve-days they are alive. Lastly, our results are stressed by the evidence that invertebrate species are as diverse as flies and worms and share a lot of genes and molecular pathways with humans. More than 90% of the domains that can be identified in human proteins are present in fruit fly and worm proteins, while 61% of fruit fly proteins and 43% of worm proteins have high sequence similarities to predicted human proteins.44 Furthermore, the gene conservation of earthworms is closer to humans than worms such as the well-known animal model Caenorhabditis 17 Figure 9. Telomerase activity in uncut, irradiated and non-irradiated (control) elegans. In fact, the bilaterian metazoan Dendrobaena veneta. The star symbol indicates the significant difference respect to the showing bilateral symmetry, are divided uncut samples. No-star symbol, P>0.05; *P<0.05; **P<0.01; ***P<0.001. into two clades: Deuterostomes and

[European Journal of Histochemistry 2018; 62:2867] [page 59] Original Paper

808-nm diode laser with a flat-top hand- piece positively photobiomodulates mitochondria activities. Photomed Laser Surg 2016;34:564-71. 10. Amaroli A, Ravera S, Parker S, Panfoli I, Benedicenti A, Benedicenti S. The protozoan Paramecium primaurelia as a non-sentient model to test laser light irradiation: the effects of an 808nm infrared laser diode on cellular respira- tion. Alt Lab Anim – ATLA 2015; 43:155-62. 11. Amaroli A, Parker S, Dorigo G, Benedicenti A, Benedicenti S. Paramecium: a promising non-animal bioassay to study the effect of 808 nm Figure 10. Expression of heat shock protein 70 (HSP70) in uncut, irradiated and non- infrared diode laser photobiomodulation. irradiated (control) Dendrobaena veneta. The star symbol indicates the significant differ- Photomed Laser Surg 2015;33:35-40. ence respect to the uncut samples. No-star symbol, P>0.05; *P<0.05; **P<0.01; ***P<0.001. 12. Amaroli A, Ravera S, Parker S, Panfoli I, Benedicenti A, Benedicenti S. Effect of 808 nm diode laser on swimming behavior, food vacuole formation and endogenous ATP production of Protostomes. Among protostomes a further Paramecium primaurelia (protozoa). subsequent distinction into two clades is References Photochemonly Photobiol 2015;91:1150-5. possible: Ecdysozoa and Lophotrochozoa. 1. Mester E, Szende B, Tota JG. Effect of 13. Amaroli A, Ravera S, Parker S, Panfoli Literature data point out that the vertebrates laser on hair growth of mice. Kiserl I, Benedicenti A, Benedicenti S. 808- and lophotrochozoa have diverged much Orvostud 1967;19:628-61. nm laser therapy with a flat-top hand- less from the ancestral genome of the 2. Parker S. Photobiomodulation: an piece photobiomodulates mitochondria urbilaterian ancestors than has the ecdyso- use activities of Paramecium primaurelia 44 overview of current concepts and zoa genome. In consequence, vertebrates understanding. In: Benedicenti A, (Protozoa). Lasers Med Sci 2016;31: and lophotrochozoa are genetically more- Benedicenti S, editors. Atlas of Laser 741-7. easily compared than vertebrates and 14. Amaroli A, Benedicenti A, Ferrando S, 45 Therapy, IV edition. Brescia, Italy; ecdysozoa. Surprisingly, D. melanogaster Teamwork media srl; 2016. p. 139-155. Gallus L, Selting W, Benedicenti S. and C. elegans are ecdysozoa, while the 3. Karu T. Primary and secondary mecha- Photobiomodulation by infrared diode Annelida, such as earthworms are lophotro- nisms of action of visible to near-IR laser: effects on intracellular calcium chozoa. radiation on cells. J Photochem concentration and nitric oxide produc- In conclusion, the 808 nm diode laser tion of Paramecium. Photochem 2 Photobiol B 1999;49:1-17. therapy, using a fluence of 64 J/cm at 1 W 4. Hamblin MR, Waynant RW, Anders J, Photobiol 2016;92:854-62. (CW) with a flat-top hand-piece, proved to editors. Mechanisms of low level light 15. Amaroli A, Gambardella C, Ferrando S, induce positive PBM. This was sustained in therapy. International Society for Optics Hanna R, Benedicenti A, Gallus L, el al. the transition from cell to the animal model. and Photonics. Proc. SPIE 6140; 2006. The effect of photobiomodulation on Three successive treatments, one every 24 h p 12. the sea urchin Paracentrotus lividus are sufficient to promote wound healing, 5. Karu TI, Afanas'eva NI. Cytochrome c (echinodermata) by using higher-flu- through effects on muscular and blood ves- oxidase as the primary photoacceptor ence on fertilization, embryogenesis, sel contraction, decrease in bacteria load, upon laser exposure of cultured cells to and larval development: an in vitro reduction of inflammatory processesNon-commercial and visible and near IR-range light. Dokl study. Photomed Laser Surg 2016; tissue degeneration. D. veneta was demon- Akad Nauk 1995;342:693-5. 35:127-35. strated to be a reliable experimental organ- 6. Carati CJ, Anderson SN, Gannon BJ, 16. Russell WMS, Burch RL. The princi- ism that satisfies the request of public opin- Piller NB. Treatment of postmastecto- ples of humane experimental technique. ion in the field of bioethics, the 3Rs princi- 16 my lymphedema with low-level laser London: Methuen; 1959. ples and the National Science Foundation therapy: a double blind, placebo-con- 17. Cressey D. The year of the worm? statement “…. All model organisms share a trolled trial. Cancer 2003;98:1114-22. Nature 2008. doi:10.1038/news.2008. few common traits: They’re inexpensive, 7. Benedicenti A, Amaroli A, Seting W, 1309. easy to care for, grow quickly and are rela- 46 Matthew A, Benedicenti S. Case studies 18. Katz PS. Model organisms in the light tively simple creatures”. on the use of a new flat-top handpiece for of evolution. Curr Biol 2016;26:R649- In acknowledgement of their genetic biomodulation in dentistry and medicine. 50. and evolutionary peculiarity, not inferior to J Laser Health Academy 2015;1-6. 19. Gurtner GC, Werner S, Barrandon Y, those of scientifically accredited models, D. 8. Selting W. Flat top hand-piece. In: Longaker MT. Wound repair and regen- veneta allows investigation of the effect of Benedicenti A, Benedicenti S, editors. eration. Nature 2008;453:15. laser therapies by multidisciplinary meth- Atlas of Laser Therapy, IV edition. 20. Ansell DM, Holden KA, Hardman MJ. ods, at various degree of complexity and Brescia, Italy: Teamwork media srl; Animal models of wound repair: are costs. 2016. p. 225-236. they cutting it? Exp Dermatol 2012; 9. Amaroli A, Ravera S, Parker S, Panfoli 21:581-5. I, Benedicenti A, Benedicenti S. An 21. Seok J, Warren HS, Cuenca AG,

[page 60] [European Journal of Histochemistry 2018; 62:2867] Original Paper

Mindrinosa MN, Bakerc HV, Xua et al. 30. Xiao N, Ge F, Edwards CA. The regen- Ruggiero K, Dunham J, Lee C, et al. Genomic responses in mouse models eration capacity of an earthworm, Phototargeting oral black-pigmented poorly mimic human inflammatory dis- Eisenia fetida, in relation to the site of bacteria. Antimicrob Agents Chemother eases. Proc Nat Academ Sci USA amputation along the body. Acta Ecol 2005;49:1391-6. 2013;110:3507-12. Sinica 2011;21:197-204. 38. Hama K. The fine structure of some 22. Schork NJ. Personalized medicine: 31. Borges TJ, Wieten L, van Herwijnen blood vessels of the earthworm Eisenia Time for one-person trials. Nature MJ, Broere F, van der Zee R, Bonorino foetida. J Biophys Biochem Cytol 2015;520:609-11. C, van Eden W. The anti-inflammatory 1960;7:717-23. 23. Lőw P, Molnár K, Kriska G. Atlas of mechanisms of Hsp70. Front Immunol 39. Coggeshall RE. A fine structural analy- animal anatomy and histology. Springer 2012;3:95. sis of the ventral nerve cord and associ- International Publishing; 2016. 32. Aarskog NK, Vedeler CA. Real-time ated sheath of Lumbricus terrestris L. J 24. Amaroli A, Ferrando S, Hanna R, quantitative polymerase chain reaction. Comp Neurol 1965;125:393-438. Gallus L, Benedicenti A, Scarfì S, et al. A new method that detects both the The photobiomodulation effect of high- peripheral myelin protein 22 duplica- 40. Augustin H, Partridge L. Invertebrate er-fluence 808-nm laser therapy with a tion in Charcot-Marie-Tooth type 1A models of age-related muscle degenera- flat-top handpiece on the wound healing disease and the peripheral myelin pro- tion. Biochim Biophys Acta 2009; of the earthworm Dendrobaena veneta: tein 22 deletion in hereditary neuropa- 1790:1084-94. a brief report. Lasers Med Sci 2018;33: thy with liability to pressure palsies. 41. Ventadour S, Attaix D. Mechanisms of 221-5. Hum Genet 2000;107:494-8. skeletal muscle atrophy. Current Opin 25. Burke JM. Wound healing in 33. Vandesompele J, De Preter K, Pattyn F, Rheumatol 2006;18:631-5. Eiseniafoetida (Oligochaeta). I - Poppe B, Van Roy N, De Paepe A, et al. 42. Asea AAA, De Maio A. Heat shock pro- Histology and 3H-thymidine radioauto- Accurate normalization of real-time teins, vol. 1. Heat shock proteins: potent graphy of the epidermis. J Exp Zool quantitative RT-PCR data by geometric mediators of inflammation and immuni- 1974;188:49-64. averaging of multiple internal control ty. Springer; Dordrecht; 2007. 26. Cholewa J, Feeney GP, O’Reilly M, genes. Genome Biol 2002;18;3: 43.only Merkling SH, Overheul GJ, van Mierlo Stürzenbaum SR, Morgan AJ, Płytycz research0034. JT, Arends D, Gilissen C, van Rij RP. B. Autofluorescence in eleocytes of 34. Kim NW, Piatyszek MA, Prowse KR, The heat shock response restricts virus some earthworm species. Folia Kim NW, Piatyszek MA, Prowse KR, et infections in Drosophila. Sci Rep 2015; Histochem Cytobiol 2006;44:65-71. al. Specific association of human telom- 5:12758. 27. Lanzavecchia G. Morphofunctional and erase activity with immortal cellsuse and 44. Rubin GM. The draft sequences: com- phylogenetic relations in helical mus- cancer. Science 1994;266:2011-5. paring species. Nature 2001;409:820-1. cles. Boll Zool 1981;48:29-40. 35. Shamirzaev B. Functional properties of 45. Raible F, Arendt D. Metazoan evolu- 28. Paniagua R, Royuela M, García- laser effects on morphology of liver, Anchuelo RM, Fraile B. Ultrastructure gall bladder and bile ducts in cholelithi- tion: some animals are more equal than of invertebrate muscle cell types. Histol asis. Med Health Sci J 2012;10:56-61. others. Current Biol 2004;14:R106-8. Histopathol 1996;11:181-201. 36. Nussbaum EL, Lilge L, Mazzulli T. 46. Arriens J. Earth’s weird and wonderful 29. Dominguez J, Edwards CA. The influence Effects of 810 nm laser irradiation on in animal models: Simple creatures reveal of vermicomposts on plant growth and vitro growth of bacteria: comparison of fundamental biological insights. pest incidence. In: Shakir Hanna SH, continuous wave and frequency modu- National Science Foundation; 2016. Mikha TlWZA, editors. Soil Zoology for lated light. Lasers Surg Med Available from: https://www.nsf.gov/ Sustainable Development in the 21st 2002;31:343-51. mobile/discoveries/disc_summ.jsp?cnt Century. Cairo; 2004. p 369-395. 37. Soukos NS, Som S, Abernethy AD, n_id=138386&org=NSF

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