Changes Of Skin Innervation During Burn Wounds Healing And Its Significance

Objective:Burn wound healing is a complex and sequencing process, which is generally divided into three interrelated/overlapping phases:inflammation, wound tissue formation, and tissue remodeling. The healing process involves interplay of epithelial-stromal-inflammatory cell-cell interactions as well as interactions between cytokines and growth factor, released by structural and immune or inflammatory cells, in order to achieve a normal recovery of the injured tissue.These mechanism have been detailed by many reports.Skin innervation plays important role in physiological wound repair, as observed during diabetic neuropathies or paraplegia, where ulcers do not repair successfully. The reason is still unknown and an explanation might be that neuropeptides or nerve growth factors may work. Few works have delineated the process of reinnervation, morphology changes of regenerated nerve fibers and impact of skin innervation on burns wound healing.Abolishment or reinforcement of skin innervation were performed in a animal experiment to observe the changes of wound healing and its healing quality, by which the role of skin innervation in burn wound healing.Meanwhile, innervation of burn wound tissue and scars were observed. With these outcomes we try to verify the rule of reinnervation during burn wound healing and throw some new light to regulate wound healing.Methods:1.Impact of innervation changes of rat on wound healingNinety wistar rats were divided into 3 groups randomly, A A(scalds+denervation,n=30),B(scalds+NGF,n=30),C(scals,n=30).Sample s of wound tissue were harvested on 7,14,21 days post burns(DPB).establishment of denervated and over-innervated rat model: the right spinal nerves from T9 to L1 were cut off in 30 rats 10 days before scalds.The denervated skin area was marked. Then each rat received one deep scald wound with diameter of 2 cm the right side of dorsal skin within the segment from T9 to L1.Each rat in group B received nerve growth fact of 1u daily via intramuscular injection for 14 days.The wound appearance,wound healing rate(WBR), wound duration were compared on 7,14,21 DPB. Histopatholgy of inflammatory cell infiltration, angiogenesis, epithelization were observed.Immunofluorescence techniques were performed to determine the NFP immune-positive nerve fibers, and nerve fiber volume fraction(NVF) were calculated for semi-quantity analysis;immunohistochemitry techniques were performed to determine the expression of collagenⅠand collagenⅢ; collagenⅠ/Ⅲratios were compared respectively. Rt-PCR technique were applied to determine the expression of VEGF mRNA,a representative marker for vessels.2.Changes of innervation during burn wound healing of human.The experiment subjects were divided into three groups:A,B,C three groups.Group A (full thickness burns wound tissues) 10 samples of 1,2,3 and 4 weeks after injury, respectively.Group B for hypertrophic scars,10 samples of proliferative hypertrophic scars(six months), respectively.Group C for the normal controls total of 10 cases were from donor sites of patients correspondingly.Each specimen was cut into three plots.One was made into a 10% neutral formalin-fixed paraffin section and stained by the modified trichrome staining method, followed by pathological observation with ordinary optical microscope.The two others were made into frozen sections after liquid nitrogen fixation and then stained by immunofluorescent staining method using anti-neurofilament protein monoclonal antibody. One of the frozen sections was 10μm thick, then ordinary fluorescent microscope was employed to observe the plane structure and the quantity of the nerve fibers.The other was 30μm thick, then the laser scanning confocal microscope was employed to observe the inner and tridimensional structure of the nerve fibers. Results:1.Impact of innervation changes of rat on wound healingWound pathology and wound healing duration:there were no difference of epithelization on 7 DPB among gourp A, B,C,and yet more inflammatory cells infiltrated in group B.The crusts of group A adhered to wound more tightly and dropped off slower than those of group B,C. The wound healing duration of group A,B,C were 23.5±2.95,18.8±1.48, 20.4±2.86 respectively.Innervation:no NFP immunoreactive nerve fibers were found in group A.NFP immunoreactive nerve fibers, with spot or cosh appearance, were found in lower part of wound tissue in group B,C on 7 DPB.Nerve fiber of group B and C,looking like spot or sprout, were increasing on 14 DPB,which further improved on 21 DBP.The NVF on 14 DBP or 21 DBP of group B is higher that of group C(p<0.05)Expression of VEGF mRNA:VEGF mRNA, increased post burns, of group A increase slowly within 14 DPB and then decreased, that of group B increased more rapidly, peaked within 7 DPB and decreased slowly. VEGF mRNA of group B is higher than that of control(p<0.05). VEGF mRNA of these three group expressed more highly than that of normal (p<0.05).CollagenⅠand collagenⅢsynthesis and collagenⅠ/Ⅲratios: collagenⅠsynthesized increasingly post burns.CollagenⅠgroup B were higher than that of group A, which is lower than that of control.There was no significant difference of collagenⅢamong group A, B and C, with an increasing trend in group A.CollagenⅠ/Ⅲratios of group A were lower than those of group C(p<0.05).2.Changes of innervation during burn wound healing of human.The CVF increased during the process of wound healing.The peak CVF was observed in scars during proliferative stage,while that of scars during maturation stage tended to normal level.Gray value and NVF of PGP9.5 positive nerve fibers were lower than those of NFP.The regenerated nerve fibers 2 WPB were sparse, short and small,which was significant lower the normal control.The skin reinnervation improved during the wound healing process and came to peak NVF in proliferative stage.Disintegration and fragmentation were observed frequently in samples from proliferative stage,which seldom occurred during mature stage.Conclusions:1.The remodeling of skin nerve fibers regenerated comprise a sequential process with increasing number, distortion shape during wound healing and then disintegrated, fractured and come to normal2.Three dimension structure of nerve fibers can get with LSCM and remodel were found during the reinnervation post burns. 3.VEGF mRNA decreases with delayed wound healing when denervated, and increases with improved wound healing while over-innervated, which means innervation has an important impact on burn wound healing.4.Over innervation would accelerate wound healing and lower inneration may improve tissue remodeling.