Innervation In Different Periods Of Granulation Tissue And Hypertrophic Scar

Objective: The deposition of collagen often exists in wound healing process after severe burn, with the result of scar formation. Reinnervation has been proved an important role in the process of wound healing and scar formation. Previous studies mostly concentrate on neuropeptides secreted by nerve fibers, but morphological changes of regenerated nerve fibers are rarely studied. We hope to investigate the rule of nerve fibers changes by comparing granulation tissues and hypertrophic scars in different periods with normal skins, then explore the function of nerve fibers in the whole process of wound healing and scar formation.Methods: The experiment subjects were divided into three groups: A, B, C three groups. Group A for granulation tissues were obtained from wounds after three degree burn. The samples were divided into 1,2,3 and 4 weeks after injury, a total of 40 cases. Group B for hypertrophic scars were obtained from post-burn or post-traumatic (1 months-23 years) patients. The samples were divided into proliferative hypertrophic scars ( six months), a total of 30 cases. Group C for the normal controls total of 20 cases were from donor sites of patients correspondingly. All patients were from the Department of Burn and Plastic Surgery of Shandong Provincial Hospital 2006-2007. 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 fluorescentmicroscope 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. Ordinary optical microscope observations: 1 week after injury there are few collagen fibers and much inflammatory infiltration. 2 weeks after injury the collagen fibers and newly born blood capillaries increase, peak at 3 weeks after injury and there is a decrease at 4 weeks after injury. The epithelium of proliferative hypertrophic scar is uniform and much thicker than the normal skin. And the thick collagen fibers has a messy distribution. The epithelium of mature hypertrophic scar is much thinner than the normal skin and the collagen fibers have a similar distribution to that of normal skin. In normal skin the adnexals are commonly observed.2. Fluorescence microscope observations: 1 week after injury nerve fibers are short, tadpole-like and hook-shaped. 2 weeks after injury nerve fibers increase slightly and are shaped like short rod, with more single-course and almost no branches. The positive area ratio of nerve fibers is much lower than the normal control(P<0.05). 3 weeks after injury nerve fibers increased significantly and are arranged parallelly or like a staggered-mesh with more branches and sprouting. The positive area ratio of nerve fibers is similar to the normal control(P > 0.05). 4 weeks after injury nerve fibers are shorter than the third week, getting together easily. The positive area ratio of nerve fibers is similar to the normal control(F > 0.05). The nerve fibers of proliferative hypertrophic scars have an irregular shape and a disordered distribution. The positive area ratio of nerve fibers is much higher than the normal control(P<0.05). The nerve fibers of mature hypertrophic scars are shaped and distributed close to the normal control and have a much lower positive area ratio than the normal control(P<0.05). The nerve fibers of normal control have uniform diameter and regular permutation. 3. Laser scanning confocal microscope observations: Nerve fibers are smallish in the early time after injury. 3 weeks after injury nerve fibers have an uneven thickness with some swelling, distortion and local fracture. 4 weeks after injury the morphology of nerve fibers is similar to 3 weeks after injury but with fewer branches. The nerve fibers of proliferative hypertrophic scars have an irregular shape and a disordered distribution with local swelling, twisting and disconnections. The nerve fibers of mature hypertrophic scars with intact inner structure and stereoscopic structure are similar to the normal skin.Conclusion:1. Laser scanning confocal microscope has an advantage in observing some changes of nerve fibers' inner structure which can not be found under ordinary optical microscope.2. The morphological changes of granulations and hypertrophic scars in different periods accompany with the innervation changes. With the wound healing, the nerve fibers decrease at first and then increase and decrease subsequently. The nerve fibers are short at first and then complicated and regulated subsequently. The changes of nerve fibers keep consistent with that of collagen fibers. It shows that the innervations of regenerated nerve fibers participate in the whole process of wound healing and scar formation, having a close relationship with the result of wound healing and the outcome of scars.