The Effects Of Local Cooling And Heating On Apoptosis And Inflammatory-related Factors In Rats With Pressure Ulcers

Objectives To compare the different effect of local cooling and heating therapy on apoptosis and inflammatory-related factors in rats with pressure ulcers(PUs),and provide the basic experimental evidence for the choice of cold and heat therapy in the treatment of clinical PUs.Methods 40 SD male rats of SPF grade were divided into four groups: Sham group,Model group,Cooling group and Heating group.We established the rat model of pressure ulcer by Ischemia reperfusion(IRI)method.The rats in the Cooling group and the Heating group were treated with cold and heat temperature respectively.At the end of the experiment,the local muscle tissue of compression was removed for the following test methods.HE staining was used to observe the skeletal muscle morphologic character;Transmission electron microscope was used to evaluate alterations of the skeletal muscle ultrastructures;Immunohistochemistry was used to detect the expression levels of Bax and Bcl-2;Immunofluorescence was used to detect the expression levels of TNF-α and IL-6;Western blot analysis was used to determine the expressions of Bax,Bcl-2,Cleaved caspase-3,Cleaved caspase-9,Cyt-c,TNF-α and IL-6;TUNEL method was used to detect the apoptotic cells in muscle tissue;Mitochondrial swelling experiment was aimed to estimate the degree of mPTP opening.Results 1 The pathological changes of rat muscle tissue showed that the muscle fibers of Sham group were normal and tightly arranged,with no obvious inflammatory cells;In the Model group,the muscle fibers were disordered,some of them were obviously broken and dissolved,and there were obvious inflammatory cells infiltration at the gap;Compared with the model group,the degree of damage was more severe in the Heating group,while was less severe in the Cooling group.2 Ultrastructural changes in rat muscle tissue showed that the muscle fbers of Sham group were correctly arranged,the sarcomere was intact,and the mitochondrial morphology was normal;In the Model group,the filaments were disordered,sarcomere contracted,mitochondrial hypertrophy,significantiy expanded,crests were reduced and vacuoles were degenerated;Compared with the model group,the degree of damage was more severe in the Heating group,while was less severe in the Cooling group.3 The results of immunohistochemistry and Western blot on the expression levels of mitochondrial apoptotic pathway related proteins in rat muscle tissue showed that,compared to the Sham group,the expression levels of Bax/Bcl-2,Cleaved caspase-3,Cleaved caspase-9 and the release of Cytochrome C from the mitochondria to the cytoplasm were significantly increased(P<0.05);Compared to the Model group,the expression levels of Bax/Bcl-2,Cleaved caspase-3,Cleaved caspase-9 and the release degree of Cytochrome C from the mitochondria to the cytoplasm in the Heating group were further increased(P<0.05),while in the Cooling group were declined(P<0.05).4 Mitochondrial swelling test showed that the extent of mitochondrial swelling was significantly increased in the Model group compared with Sham group(P<0.05);Compared with the Model group,the extent of mitochondrial swelling in the Heating group was further increased(P<0.05),while in the Cooling group was decreased(P<0.05).5 TUNEL results showed that the number of positive cells in the Model group was significantly higher than that in the Sham group(P<0.05);Compared with the Model group,the apoptotic nuclei in the Heatingt group were increased further(P<0.05),while in the Cooling group were decreased(P<0.05).5 The results of Immunofluorescence and Western blot on the expression levels of TNF-α and IL-6 in rat muscle tissue showed that,compared to the Sham group,the expression levels of TNF-α and IL-6 were significantly increased(P<0.05);Compared to the Model group,the expression levels of TNF-α and IL-6 in the Heating group were further increased(P<0.05),while in the Cooling group were declined(P<0.05).Conclusions 1 The muscle tissue damage was arised in the early-stage of PUs in rat,the inflammatory factors and mitochondrial apoptotic pathway were involved in the formation of early-stage PUs.2 Local heating can increase the degree of compressed tissue,while local cooling can decrease the damage of compressed tissue,therefore,heat therapy may not be suitable for the clinical treating and caring in the early-stage of PUs,while cold therapy may be more appropriate.