The earthworm Dendrobaena veneta (Annelida): A new experimental-organism for photobiomodulation and wound healing

Abstract

Photobiomodulation (PBM) is a manipulation of cellular behavior using non-ablative low intensity light sources. This manipulation triggers a cascade of metabolic effects and physiological changes resulting in improved tissue repair, of benefit in the treatment of tissue injury, degenerative or autoimmune diseases. PBM has witnessed an exponential increase in both clinical instrument technology and applications. It is therefore of benefit to find reliable experimental models to test the burgeoning laser technology for medical applications. In our work, we proposed the earthworm Dendrobaena veneta for the study of non-ablative laser-light effects on wound healing. In our preliminary work, D. veneta has been shown to be positively affected by PBM. New tests using D. veneta were set up to evaluate the effectiveness of a chosen 808 nm-64 J/cm2-1W-CW laser therapy using the AB2799 hand-piece with flat-top bean profile, on the wound healing process of the earthworm. Effective outcome was assimilated through examining the macroscopic, histological, and molecular changes on the irradiated posterior-segment of excised-earthworms with respect to controls. Three successive treatments, one every 24 hours, were concluded as sufficient to promote the wound healing, by effects on muscular and blood vessel contraction, decrement of bacteria load, reduction of inflammatory processes and tissue degeneration. D. veneta was demonstrated to be a reliable experimental organism that meets well the 3Rs principles and the National Science Foundation statement. Through their genetic and evolutionary peculiarity, comparable to those of scientifically accredited models, D. veneta allows the effect of laser therapies by multidisciplinary methods, at various degree of complexity and costs to be investigated.

Keywords: Heat Shock Protein; Low Level Laser Therapy; anti-inflammatory; cell proliferation.; laser irradiation; scarring; tissue repair.

Figure 1.

Effect of the laser irradiation on the temperature of Dendrobaena veneta excised side. The temperature was measured before (control) and after (irradiated) the laser irradiation.

Figure 2.

Bacterial load analysis in non-irradiated (control) and irradiated excised side of Dendrobaena veneta. A= bacterial colonies grown for 24 h on Tryptic Soy Agar. B= image analysis of the area of bacterial colonies grown for 24 h on Tryptic Soy Agar. C= spectrophotometric analysis of the bacterial colonies grown for 24h on Tryptic Soy Agar. D= spectrophotometric analysis of bacterial colonies grown for 3 h, 6 h and 24 h in LB broth. O.D.= optical density. No-star symbol= P>0.05; *= P<0.05; **= P<0.01; ***= P<0.001.

Figure 3.

Dendrobaena veneta survival rate expressed as a percentage, in non-irradiated (control) and irradiated (3 irradiation) excised site. No-star symbol= P>0.05; *= P<0.05; **= P<0.01; ***= P<0.001.

Figure 4.

Effect of the laser irradiation on the wound healing of Dendrobaena veneta 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 organism immediately after the first irradiation (0 h). C=non-irradiated (control) earthworm 24 h after the excision and D, earthworm 24 h after the first irradiation. E=nonirradiated (control) earthworm 48 h after the excision and F, earthworm 48h after the first irradiation and 24 h after the second irradiation. G= non-irradiated earthworm 72 h after the excision and H, earthworm 72 h after the first irradiation and 48 h and 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 and third irradiation, respectively. at= alimentary tract; cf= coelomic fluid. Bars= 2.3 mm.

Figure 5.

Histological section of 30 posterior excised segments of Dendrobaena veneta. Masson’s Trichrome stain (A-D) or Hematoxylin and Eosin stain (E, F). A, C, E= non-irradiated (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, 2, 3 irradiations, respectively. *=everted alimentary tract wall. Ce=celom; Il= intestinal lumen; Mu= muscular tissue. Bars, 300 m.

Figure 6.

Histological section of Dendrobaena veneta excised side. Masson’s Trichrome stain, 24 h after excision, in non-irradiated (control) (A) and irradiated, 1 irradiation, (B) earthworms. Hematoxylin and Eosin staining, 48 h after excision, in non-irradiated (control) (C) and irradiated, 2 irradiations, (D) earthworms. A-C-D, bars, 100 m. B, bars 20 μm. Ce= celom; Mu= muscular tissue; ▲= blood vessel lumen.

Figure 7.

Histological section of Dendrobaena veneta excised side. Masson’s Trichrome stain. A,C,E) Non-irradiated (control) samples, 24, 48 and 72 h from the excision, respectively. 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 tissue; MuL, longitudinal muscle tissue; Ct, connective tissue. Scale bars: 5 m.

Figure 8.

Histological section of Dendrobaena veneta excised side. The Alexa 488-conjugated lectin wheat germ agglutinin (WGA) binds (green fluorescence, white arrows) the N-acetylglucosamine and N-acetylneraminic acids. The DNA is point out by DAPI fluorescence staining (blue fluorescence). A) The WGA is positive in the cytoplasm of scattered cells in the peritoneum of both non-irradiated and irradiated samples. B,D,F) Nonirradiated (control) samples 24, 48 and 72 h after the excision. C,F) Irradiated samples, 24 and 48 h from the excision or, on the other hand after 1 and 2 irradiations, respectively. Ce,celom; S, septum; Ep, epidermal tissue; MuC, circular muscle tissue; MuL, longitudinal muscle tissue. Scale bars: 50 m.

Figure 9.

Telomerase activity in uncut, irradiated and non-irradiated (control) Dendrobaena veneta. The star symbol indicates the significant difference respect to the uncut samples. No-star symbol, P>0.05; *P<0.05; **P<0.01; ***P<0.001.

Figure 10.

Expression of heat shock protein 70 (HSP70) in uncut, irradiated and nonirradiated (control) Dendrobaena veneta. The star symbol indicates the significant difference respect to the uncut samples. No-star symbol, P>0.05; *P<0.05; **P<0.01; ***P<0.001.