Effects Of Persistent And Intermitten Hypoxia-controlled Tissue Expansion On Dermal Histological Changes

Skin and soft tissue expansion is an effective method for the treatment of soft tissue defects and malformations.During the expansion process,the flap produces a series of changes,including the proliferation and apoptosis of the synthesis and degradation of extracellular matrix,the formation and degeneration of new blood vessels,etc.The oxygen supply of the tissue plays a key role in such changes.However,in different ways of expansion,there are obvious differences in the degree of hypoxia and the duration of hypoxia.In this way,the biological characteristics of fibroblast proliferation and apoptosis,collagen synthesis and decomposition,generation and degeneration of new blood vessels are changed,which directly affect the quality of the expanded skin.The first part of this paper introduces the experimental method of intermittent hypoxia and continuous hypoxia skin expansion model in rats.Tissue hypoxia detection reagent and percutaneous oxygen partial pressure were used to determine the hypoxia state of the tissue.HE staining is used to determine the effect of tissue expansion.It is hoped to provide a mature and stable model of anoxic expansion for the basic research of expansion.In the second part of this paper,the histological changes of dermis were studied under the conditions of intermittent hypoxia and continuous hypoxia.Through HE staining,the changes of dermal structure were observed under the microscope,to measure the change of dermis thickness and collagen content in each group;PCAN staining and Tunnel staining were used to detect the proliferation and apoptosis of dermal fibroblasts;CD31staining to detect the derma vascular density.In order to understand whether the expansion under different hypoxia conditions can effectively induce the reconstruction of the dermal tissue,and have a detrimental effect on the tissue.It provides experimental basis for the clinical search for suitable expansion mode.Part 1 Induction of intermittent and persistent hypoxia during skin expansion in a rat modelObjective:To establish a reliable rat model of intermitten and persistent hypoxia during skin expansion and to evaluate the relevant histological changes.Methods:Twenty four Sprague-Dawley rats,weighing 200-250 gram,were divide into 2groups:Persistent hypoxia group?PH?,Intermittent hypoxia group?IH?,with 12 rats in each group.A skin and soft tissue expander of 10 ml was subcutaneously implanted in the back region of each rat.The expanders in PH group received saline injection daily and the transcutaneous oxygen pressure?tcpO₂?of the expanded skin was monitored until the tcpO₂ reached 1-3 mmHg at the end of each injection.Such daily procedure continued until the sum of the volume of injected saline reached 70 mL for each expander that maintained 1-3 mmHg tcpO₂ in the expanded skin.For IH group,saline was injected into the expanders until the tcpO₂ of the expanded skin reached<3 mmHg and the changes in tcpO₂ of the expanding skin were monitored.When the values of tcpO₂ were recovered to the normal,pre-injection levels,the saline injections were repeated.This procedure continued until the total volume of the saline in each expander reached 70 mL.The skin samples in the expanded group and control group were collected,fixed,embedded in paraffin,sectioned and stained with HE staining.Changes in skin structure were observed under microscope.Tissue hypoxia state was quantitatively determined with immunofluorescence staining of a hypoxia-specific probe(Hypoxyprob^TM-1)in the pre-treated skin tissues of the three experimental groups.Results:Compared with normal skin,the epidermal layer of the expanded region was significantly thickened in IH and PH groups.More dermal hypoxic fibroblasts was found PH group than that of IH group.Conclusion:Skin and soft tissue expansion in the back region of rats can provide a satisfactory model for skin expansion research during different hypoxia conditions.Part 2 Effects of Persistent and Intermitten hypoxia-controlled tissue expansion on dermal histological changesObjective:To explore the dermal histological changes under persistent tissue hypoxia expansion and intermitten tissue hypoxia expansion.Methods:Collection of persistent hypoxia expansion,intermitten hypoxia expansion and normal skin as specimens.Put the specimen into formalin,and then take it to make paraffin section to part into HE dyeing,Masson dyeing and immunity histochemistry of PCAN,TUNEL and CD31 dyeing.Observe routine HE dyeing microtome section,observe the histology change of each group with microscope and measure the thickness of dermal with micro-ruler,Stochastically selecting 10 pieces of campus visualis of HE dyeing microtome section amplified 100 times of experiment;Masson dyeing microtome section with microscope,by quantitative analyzing the average gradation of the dyeing example with pathology image analysis system,quantificating the content of collagen fibers in each groups.The number of proliferating fibroblast cells in dermal layer was counted using immumohistochemical staining of PCNA;The number of apoptosis fibroblast cells in dermal layer was counted using immumohistochemical staining of TUNEL;Measure the content of collagen fibers in each group;Observe CD31 dyeing microtome section with microscope,compare the quantitative change of micrangium in dermal in each group.Result:The thickness of dermal of IH group is 500.2±22.57?m,the thickness of derma of PH group is 464.3±27.23?m,the thickness of dermal of normal group is 759.4±27.23?m.The dermal of normal group were significantly thicker than IH group and the PH group.The IH group was significantly thicker than the PH group,and the difference was statistically significant?P<0.05?.By topography quantitative analysis,the meangradation of normal group is 199769±6027 pixels;the meangradation of IH group is257557±4648 pixels;the meangradation of PH group is 220401±7298 pixels?P<0.05?.The IH group was significantly higher than the PH group,and the difference was statistically significant?P<0.05?.The PCNA positive cells in PH group decreased compared with IH group,and the difference was statistically significant?P<0.05?.The TUNEL positive cells in IH group decreased compared with PH group,But there was no statistical difference in the result?P>0.05?.The quant of micrangium in each campus of IH group is 22.87±1.13,the quant of micrangium in each campus of PH group is 13.57±0.43?P<0.01?.Conclusions:1.The proliferation or apoptosis fibroblast of expansion had close correlation with the maintenance period and the stimulus intensity of tissue hypoxia.2.The intermittent hypoxia expansion ways can effectively improved the synthesis and secretion of collogen than thehypoxia expansion flaps.3.The intermittent hypoxia expansion ways can effectively improved vascular density in the expansion skin flaps than persistent hypoxia expansion ways.