Therapeutic Efficacy Of Early Low Level Laser(Light)Therapy On The Zones Of Stasis In Burns

Background:The issue of progression of injury in the zone of stasis in burn often contributes to both greater burn surface area and burn depth.This larger,deeper wound has multiple local and systemic consequences that increase complications and morbidity.For the large surface area burn victims,burn wound progression can aggravate patient's condition,increases the difficulty of treatment,and prolongs the hospitalization time.For the small surface area burn victims,burn wound progression can exacerbate scar formation,affecting the appearance and function.Thus it is important to develop a novel therapy to halt the progression of injury in the zone of stasis.Low level laser(light)therapy(LLLT),also known as photobiomodulation,which refers to the use of low-energy red light and infrared light sources(wavelengths ranging from 600 to 1000 nm)to illuminate human tissue,resulting in a series of photobiological stimulating effect.Numerous studies have shown that LLLT has achieved significant efficacy in the treatment of acute and chronic wounds and no adverse reactions have been observed.LLLT can reduce local inflammatory,edema and pain,stimulate proliferation and migration of fibroblasts,keratinocytes,vascular endothelial cells and osteoblasts.LLLT aslo can promote collagen synthesis,promote capillary angiogenesis,and promote the synthesis and release of growth factors.However,the current role of LLLT on the progressive necrosis of the zone of stasis in the burn wounds has not been clearly reported.Therefore,the zone of stasis model of rats was established in this study first and then we explored the therapeutic effect of early local LLLT on the zone of stasis.Part 1: The construction of the zone of stasis modelObjective: To establish and verify the zone of stasis model in burnsMethod: The rats were divided into Control Group(CG,n=10),Model Group 1h(MG 1h,n=10),Model Group 24h(MG 24 h,n=10),Model Group 96h(MG 96 h,n=10).The rats were anesthetized with 1% sodium pentobarbital.Their dorsal skin was shaved using an electric clipper,after which a commercial depilatory cream was applied to remove all remaining hair.The zone of stasis model was established according to brass comb burn model invented by Regas FC and Ehrlich HP.For MG,A comb-shaped brass probe with teeth spaced at 0.5-cm intervals was used to establish the zone of stasis model.After a 15 minutes incubation in 98 oC-100 oC boiling water to ensure even heating,the brass probe was placed on the burn site and held for 10 s without additional pressure.Two comb burns were created on each rat,one on each side.The burn sites which was directly contacted were be regarded as the zones of necrosis,while the interspaces are not directly injured,The interspaces thus represent the zone of stasis.For CG,the brass comb was put in the 23 oC-25 oC water for 15 minutes and then was placed on the burn site and held for 10 s without additional pressure.No dressings or topical treatment were applied to any of the animals.Wounds were observed daily for evidence of necrosis in the unburned interspaces and image processing software was used to calculate the percentage of necrosis in the stasis area.HMGB1 immunohistochemistry was applied to calculate the density of necrotic cells.HE staining,Masson staining and skin ultrasound were used to evaluate the depth of burn in the zone of stasis.Doppler angiography analyse was used to investigate blood flow changes in the zone of stasis.Result: At 1 h,24 h and 96 h after injury,gross observation showed that the necrotic tissue in the zone of stasis of MG rats gradually increased,and adjacent necrotic areas had a tendency to fuse.The percentage of necrosis in the stasis area for CG,MG 1 h,MG 24 h and MG 96 h calculated by the image processing software were 0%,2%,30%,64%,respectly,and these results has shown the percentage of necrosis in the stasis area increased continuously,P<0.05;the density of necrotic cells abtained by HMGB1 immunohistochemistry were 1.2�0.3(Per visual fields),14.2�2.0(Per visual fields),58.0�8.5(Per visual fields),81.3�13.9(Per visual fields),the results has shown that the density of necrotic cells in the stasis area increased continuously,P<0.05.HE staining and Massion staining showed that,as time goes on,the epidermis gradually became necrotic,the dermal edema was aggravated,the collagen was gradually degenerated and necrotic,the inflammatory cells were accumulated,and the red blood cell gradually appear in the microvessels.The results of high-frequency ultrasound showed that there was no significant change in epidermal thickness among all groups.P>0.05;the epidermal density increased continuously,P<0.05;the overall skin thickness increased continuously,P<0.05.There was a significant difference in the overall skin density among CG,MG 1 h,MG 24 h,P<0.05.NO significant difference was found between MG 24 h and MG 96 h.The results of Ultrasound Doppler angiography showed that the skin blood flow for CG,MG 1 h,MG 24 h and MG 96 h were 13.6 � 1.0 ml LD / min / 100 g,12.1� 0.6 ml LD / min / 100 g,9.3 � 0.5 ml LD/min/100 g,3.7�0.5ml LD/min/100 g,and blood flow value decreased continuously,P<0.05.Conclusion:1.The zone of stasis model was successfully constructed by Brass comb method2.The area of necrosis,burn depth,the cell necrosis density in the zone of stasis in this study had been increasing continuously within 96 h.And the blood flow in the zone of stasis in this study had been decreasing continuously within 96 h.Part 2:Explore therapeutic efficacy of early photobiomodulation therapy on the zones of stasis in burnsObjective: Explore therapeutic efficacy of early photobiomodulation therapy on the zones of stasis in burnsMethod: The rats were randomly divided into control groups(CG)and laser groups(LG),15 rats in each group.The zone of stasis was formed by method used in the part 1 resulting in 8 rectangular burns separated by 6 unburned interspaces in each side.The left side was laser wound(LW),while the right side contained the shielded wound(SW).The LW in the LG were immediately subjected to photobiomodulation therapy,followed by once-daily 30-min local photobiomodulation therapy sessions.Skin ultrasound and Doppler angiography analyses were used to evaluate the statuses of the zones of stasis at 1,24,and 96 h after injury and calculate the proportions of necrosis in these areas.Harvested burn wound tissue was subjected to hematoxylin-eosin staining and HMGB1,caspase 3,and thrombomodulin(TM)immunohistochemistry,and the contents of NO and TNF-? were measured in stasis tissue.Results: Gross observation: at 96 h after injury,the proportion of necrosis in differed significantly to others.The percentage of necrosis in the stasis area in the LW of the LG was significantly reduced at 96 h after injury,compared to the other types of wounds.HE staining reaults: Less epidermal necrosis and exfoliation,less dermal edema,less aggregation of inflammatory cells,and a small amount of erythrocyte deposition in microvessels were observed,other wounds performed similarly.The results of high-frequency ultrasound showed the stasis of LWs of LG exhibited a lower epidermal density and overall skin thickness and higher overall skin density compared to the CG wounds(p <0.05)at 96 h after injury.The results of Ultrasound Doppler angiography showed that the stasis of LWs of LG exhibited a significantly higher skin blood flow relative to others at 96 h(p <0.05).TM,HMGB1,and caspase 3 immunohistochemistry revealed significantly lower positive staining rates in the LW of LG rats relative to other burn wounds at 96 h(p <0.05 for all).The NO content in the stasis area was significantly higher in the LW of LG,compared to other wounds,at 24 and 96 h after injury(p <0.05).The TNF-? level was significantly lower in the LW of LG than in other wounds at 96 h(p <0.05).In addition,the study found there were no significant differences between the CG and LG's SW zones of stasis(p >0.05)on the basis of the result of gross observation,HE staining,high-frequency ultrasound,Ultrasound Doppler angiography,TM,HMGB1,and caspase 3 immunohistochemistry and TNF-? measurement(p >0.05).Only one indicator had statistical difference which was the result of NO measurement.The NO content was significantly higher in the SW of LG,compared to the bilateral wounds of the CG at 96 h after injury(p <0.05).Conclusion:1.Early,local photobiomodulation therapy can effectively ameliorate injury progression in the zone of stasis.However,these beneficial effects are limited to the directly irradiated sites.2.The therapeutic efficacy of early photobiomodulation therapy on the zones of stasis in burns may be associated with that it can improve the blood flow in the zones of stasis,reducing local inflammation,reducing local apoptosis and cell necrosis.