Fig 1.
Positioning of the rat and photobiomodulation (PBM) therapy with 810 nm diode laser applied on wound at the dorsal surface of rat without contact.
Scale showing the wound diameter size (1.5 cm).
Table 1.
List of laser parameters of continuous wave.
Table 2.
List of laser parameters of Pulse frequency 10 Hz.
Table 3.
List of laser parameters of Pulse frequency 100 Hz.
Fig 2.
Photomicrographs showing progression during healing of the full thickness dermal wounds in 810 nm LLLT irradiated groups as compared to non-irradiated control and silver sulfadiazine (SSD) ointment (reference care) treated wounds in immunosuppressed rats.
Fig 3.
Effect of 810 nm laser on wound contraction rate (% change) in immunosuppressed rats.
At days 4 through 8, the 810 nm LLLT (40 mw/ cm2, 22.6 J/ cm2) 10 Hz group shows a smaller wound area than CW and 100 Hz groups compared to the control. Values are mean ± SEM, n = 6 animals per group. *p < 0.05 compared to the non-irradiated control.
Fig 4.
Effect of 810 nm LLLT for 7 days on pro-healing biochemical markers on dermal wound healing in immunosuppressed rats.
Values are mean ± SEM, n = 6 animals per group. *p < 0.05 compared to the non-irradiated control.
Fig 5.
Morphology of the wound skin on eighth day post-wounding in non-irradiated control, 810 nm LLLT irradiated and silver sulfadiazine (SSD) ointment (reference care) treated wounds in immunosuppressed rats.
Lower (10X) and higher (40X) magnification showing widespread regeneration of epidermal cells with mature differentiation, greater amount of fibroblast cells, compact collagen deposition, less infiltration of inflammatory cells, new blood vessel formation (angiogenesis) and complete re-epithelialization in 810 nm LLLT irradiated groups, among LLLT irradiated groups these finding was more prominent in PW 10 Hz frequency treated wound. However, non-irradiated control and SSD treatment showed very less epithelial cell regeneration, reduced level of collagen and infiltration of inflammatory cells with less fibroblast. Arrow marked the epithelial migration towards the wound tissue, (E) denotes the skin epithelium and the dotted line illustrate the epithelial margin among groups. Scale bar, 10 μm.
Fig 6.
Cytochrome c oxidase (CCO, mitochondrial complex IV) activity, TNF-α and ATP levels in non-irradiated control, 810 nm LLLT irradiated and silver sulfadiazine (SSD) ointment (reference care) treated wounds after 7 days post-wounding in immunosuppressed rats.
Values are mean ± SEM, n = 6 animals per group. *p < 0.05 compared to the non-irradiated control.
Fig 7.
Protein expression of MMP-2 and 9 by gelatin zymography in 810 nm laser irradiated and non-irradiated control wound tissues after 7 days post-wounding.
Densitometric analysis using Fiji software. Change in expression expressed as net intensity (% control). Values are mean ± SEM, n = 6 animals per group. *p < 0.05 compared to the non-irradiated control.
Fig 8.
Western blotting assay of the growth factors, extra cellular matrix (ECM), cellular and nuclear protein viz. vascular fibroblast growth factor receptor-1 (FGFR-1), fibronectin, vascular endothelial growth factor (VEGF), smooth muscle α actin (α-SM actin), heat shock protein (HSP)-90, nuclear factor (NF)-kB, hypoxia inducible factor (HIF-1α) and transforming growth factor (TGF-β2) in non-irradiated control, 810 nm LLLT irradiated and silver sulfadiazine (SSD) ointment (reference care) treated wounds in immunosuppressed rats after 7 days post-wounding.
Densitometric analysis using Fiji software. Change in expression expressed as net intensity (% control). Values are mean ± SEM, n = 6 animals per group. *p < 0.05 compared to the non-irradiated control.
Fig 9.
Mechanism of action of photobiomodulatory effects of 10 Hz-pulsed wave 810 nm (NIR) laser showing anti-inflammatory effects, an enhance cellular proliferation, neo-vascularization, ECM accumulation in conjunction with tissue remodeling and bioenergetics activation, which collectively augments dermal wound healing in immunosuppressed rats.