Negative Pressure Combined Topical Oxygen Therapy For Rat Dorsal Deep Partial Thickness Burn Wound Progression

Background: Bunrs is a dynamic injury. Burn wound is not permanent, under multiplefactors it will be deeper and wider. The nature of this burn wound progression is death ofcell in the zone of stasis, main causes of cell death include inflammatory cell infiltrates,blood flow changes, and oxygen derived free radicals. Traditionally, death of cell wasdivided into two kinds, apoptosis and oncosis, however, the type II programmed celldeath, autophagy, has not been studied in burn wound yet. Negative pressure woundtherapy (NPWT) has been widely used over diversed acute and chronic wound in peaceand war, and its mechanism has been gradually disclosed either, include edema remove,exudation draining and improve local blood flow. The effect of topical oxygen therapy(TOT) for wound is positive, although it mechanism needs further elucidate. NPWT andTOT have been alone used in burn wound, and our research team member Zhang ZP andLiu HP have proved that combined theses two therapies could improve ischemic woundhealing successfully, but their mechanisms are still not well known. This experimentaimed to build a reliable deep partial thickness burn wound, and explored the cause ofcell death in the zone of stasis, and then used negative pressure combined topical oxygentherapy for burn wound to further elucidate their mechanisms and synergistic effect ofwound treatment and provided evidence for clinical use. This experiment had5parts asbelow. Methods and ResultsPart I: Based on reference, we customed made a brass comb and heated it in boilingwater for5min, blotted dry, and then quickly placed on the shaved and depilated dorsumof rat, and held for12s,16s,20s,24s,28s without any pressure, and then a burn woundwith3interspaces were made. We used image analysis and hematoxylin and eosinstaining to observe the change of interspace area and burn wound depth, and combinedwith gross and microscope observation to analyse. Results: Brass comb burn with20scould make a deep partial thickness burn wound, and interspaces would have burn woundprogression, which is met the standard of the zone of stasis animal model.Part II: Immunohistochemistry was used for analysis the distribution of autophagicmarker Beclin1and apoptotic cell by TUNEL in the zone of stasis. Western blotting wasused for evaluation Bcl-2and Bax, which were dual-regulators of autophagy andapoptosis. Autophagy was mainly strongly expressed in hair follicle epithelium from2hours postburn, and rapidly arrived peak at12hours postburn then gradually and gentlydeclined. Differently, apoptosis was mainly located in stratum epidermis and lightlyexpressed until12hours postburn, and then slowly increased and obvious staining wasobserved at48hours postburn. These results suggest that early treatment for burn woundprogression should take autophagy int consideration.Part III: Negative pressure combined topical oxygen for rat dorsal deep partial thicknessburn wound. There are negative pressure group(NP, continuous negative pressure75mmHg,4hrs), topical oxygen group (TO,40%±5%humidifying pure oxygen,90min),negative pressure combined topical oxygen group (NO, continuous negative pressure75mmHg and40%±5%humidifying pure oxygen at the same time,4hrs), and controlgroup (CT, dressing by single layer oil gause). All is change fresh dressing per two days.Biopsies were taken before treatment, and after treatment at2,4,6and8days. NOgroup’s interspace area decreased the least and burn wound depth declined the most(P<0.05). Except CT group, between the treatments from2d to8d, comparison of otherthree groups’interspaces change, the difference was not statistically significant. Thissuggested that different groups’effect of limiting the zone of stasis progression was mainly in2days.Part VI: Observation of oxidative stress index of rat deep partial thickness burn woundinterspace after four different treatments. The same groups as Part III, samples wereharvested from interspace before treatment and after treatment at2,4and6days, andrelated kits were used to analyse the activity of superoxide dismutase (SOD) and catalase(CAT) and the level of malondialdehyde (MDA). NO group’s activity of SOD and CATwere promoted and level of MDA was lower at each time point, comparison in groups,the statistically difference is significant (P<0.05). NO group could obviously attenuatethe oxidative stress response of deep partial thickness burn wound progression.Part V: The same groups as Part III, samples were harvested from interspace beforetreatment and after treatment for2,4and6days, qRT-PCR was used for semiquantitative NOS, XOD and NOX mRNA expression. NO group’s NOS, XOD and NOXmRNA expression decreased from treatment began(P<0.05), indicated that its protectionof the zone of stasis from oxidative stress was better than other groups.ConclusionBrass comb burn model is a reliable and repeative animal model for burn woundprogression research. After heated brass comb in boiling water for5minutes, and thenplaced it on skin for20seconds could make a deep partial thickness burn wound. Thiswound would go deeper and wider, and unburn skin of interspace would be progressivenecrosis in48hours. Autophagy and apoptosis play roles in burn wound progression, andtheir relative importance would change along with time. Autophagy occured earlier andmainly in dermis layer hair follicle, and apoptosis occurred later and most in epidermis.Negative pressure combined topical oxygen could obviously limit burn woundprogression, considering autophagy occurs in2hours postburn, and its applied timeshould be controlled in2-3hours postburn and maintained for48hours at least. Negativepressure therapy could clear necrotic tissues, edema remove and improve blood flow,these are beneficial for oxygen diffusion. Two therapy combine together could meetmutual complementarity. NO for deep partial thickness burn wound could promote theactivity of SOD and CAT, and decrease the level of MDA, and decline the mRNA expression of NOS, XOD and NOX. Attenuate oxidative stress and limit burn woundprogression, these might be synergetic mechanism of negative pressure combined topicaloxygen.