Experimental Study About Healing Characteristics Of Full-thickness Skin Wounds In Diabetic Mice

Part 1 Comparative Study on Skin Wound Healing in Two Types of Diabetic Mouse ModelObjective To compare the merits and the demerits of both diabetic mous e models, streptozotocin (STZ)-induced and spontaneous genetically induced mo use, which have been widely used recently, and to provide the guidance for fu rther studies on diabetic wound healing research, the time of skin wound heali ng and the gene expression levels of VEGF-A、Col-Iα mRNA were investigated in current study.Methods 1) 24 four-week-old ICR male mice were randomly divided into two groups,12 for the control group (ND group) and 12 for the high-fat diet +STZ group (STZ group), respectively fed with normal diet or high fat diet for 4 weeks. The mice of STZ group were given twice STZ injection at 80mg /kg, intraperitoneally to induce diabetes. Mice were wounded after 4 more wee ks fed with same diet according to grouping.2) 12 C57BL/KsJ (db/db) mice ( db/db group) and C57BL/KsJ (db/+) mice (db/+ group),10-11 weeks old with half male and half female, were also employed in the studies.3) Each mouse of 4 groups was created a dorsal full-thickness skin defects wound under isoflu rane inhalation anesthesia. At the 0,7,10,14,17,21,24, and 28 days after wounding, wound dressing replacement and wound photography were conducted and wound tissues were harvested on day 7 and 14 after wounding. The rati o of wound healing was measured with NIH ImageJ, and the expression of V EGF-A and Col-Iα mRNA in wound tissues was detected with real time-PCR.Results Compared with the control groups (ND group and db/+group), th e wound healing progress in both groups of diabetic mouse (STZ group and d b/db group) were delayed, while the wound healing ratio of both diabetic grou ps at the day 7,10, and 14 after wounding expressed significantly different (P <0.05). Between the two diabetic groups, the wound area enlarged at day 3 aft er wound. However, compared with the STZ group, the wound healing progres s of db/db group significantly delayed after day 3. The length of re-epithelializ ation of the diabetic mice (STZ group and db/db group) was lower than those of the control groups (ND group and db/+group), especially at day 7 after wounding (P<0.05). But, at the day 14 after wounding, there was no statistical significance for both diabetic groups compared with the control groups, even t he numerical value of re-epithelialization was still lower. The expressions of ge nes related with wound healing showed, that both VEGF-A and Col-la mRNA in the wound tissues of two diabetic groups were lower compare with those of control groups (P<0.05). However, there was no statistical significance at 14 days after wounding, even the numerical value was still smaller than that of t he control groups.Conclusion Both diabetic full-thickness skin defects wound models are typ ically featured by delayed wound healing and lower expression of the wound h ealing related mRNA. But, take account of the extent of wound healing, diabet ic db/db mouse caused by spontaneous genetic mutations are more suitable for chronic wound research.Part 2 Study on Healing Characteristics of Full-thickness Skin Wounds in Diabetic db/db MiceObjective On the basis of the previous research, to further explore the pat hological and physiological characteristics of full-thickness skin defect during w ound healing in diabetes db/db mice with spontaneous DNA mutation and to u nderstand the main influence factors of healing delays, and to provide the theo retic foundation for the further study of diabetic chronic wound healing mecha nism and therapy.Methods 12 spontaneous mutation diabetic C57BL/KsJ (db/db) mice (db/d b group) and 12 C57BL/KsJ (db/+) mice (db/+group),10-11 weeks old with half male and half female, were also employed in the studies. The db/db mic e were grouped in the experimental group (hereinafter referred to as db/db) a nd db/+ mice were grouped in the control group (hereinafter referred to as db/ +). A 1 cmxl cm of full-thickness skin wound on the dorsal skin was created under anesthesia. At the 0,7,10,14,17,21,24, and 28 days after skin wou nding, dressing replacement and wound photography were conducted, and woun d tissues were harvested on day 7 and 14 after wounding. The ratio of wound healing was measured with NIH ImageJ, and the expression of VEGF-A, VE GFR2, IL-6, CXCR4, CXCL12, Col-la, a-SMA mRNA in wound tissues was detected with real time-PCR. In addition, the degree of wound tissue neovascul arization was observed by carrying out the CD31 immunohistochemistry.Results Compared with the control group (db/+group), the wound healing in diabetes (db/db group) was delayed than the control group (db/+group). T he former was basically healed in 17 days after wounding while the later was delayed to 28 days after wounding. For wound healing rate, it was significantl y reduced (P<0.05) in diabetes db/db mice at the 7,10 and 14 days while the length of newly grown epithelia was also significantly declined at the day 7a nd 14 after wounding (day 7, P<0.05; day 14, P<0.01). Real-time quantitati ve PCR results showed that wound neovascularization-related factors VEGF-A a nd VEGFR2 mRNA’s relative expressions in diabetes db/db mice were significa ntly reduced (P<0.05) at the day 7 and 14 after wounding; and inflammatory f actor IL-6 mRNA’s relative expression was also significantly reduced (P<0.05) at same time points; but chemokine CXCL12 and its receptor CXCR4 mRNA’s relative expressions had a downtrend. However, it had no statistical significan ce (P>0.05) compared with normal control db/+ mice. Extracellular matrix (EC M) ingredient Col-Iα mRNA’s relative expression was significantly decreased (P <0.05) at the day 7 after injuring. Even though a-SMA mRNA was lower than that of db/+ mice of normal control group at the day 7 and 14 after injuring, it had no statistical significance (P>0.05). Wound histological sections of CD3 1 staining suggested that the wound neovascularization in diabetes db/db mice was significantly lower than that of db/+ mice (P>0.05) of normal control grou p on the day 7 after injuring.Conclusion The wound model of full-thickness skin defect in diabetes db/ db mice with spontaneous gene mutation were observably featured by delayed wound healing and showed some characteristics of depressed partial inflammato ry reactions, decreased inflammatory cell infiltration and epidermis proliferation, ECM deposition, and suppressed neovascularization, which were similar with c linical patients. It can be considered as a reliable animal model for diabetic ch ronic wound healing studies.