To design a mouse model of skin flap ischemia-reperfusion which is suitable and reproducible, clarify the mechanism of ischemia reperfusion injury (IRI) of the skin flap, and elaborate the relationship between the ischemia time, reperfusion blood flow and the degree of IRI.

A total of 162 SPF grade healthy male ICR mice were selected as the experimental subjects. (1)In the experiment one, 90 IRI mice were respectively drawn into 3 groups (n=30): thoracic skin flap group, dorsal skin flap group and epigastric skin flap group, and then the mice in each group were divided into 6 groups (n=5), Ⅰ, Ⅱ, Ⅲ, Ⅵ, Ⅴ, Ⅵ group according to the time of the vessel pedicled clamped(time of ischemia), the corresponding ischemia time respectively was 0, 1.5, 3.0, 5.0, 8.0, 10.0 h. The 3.0 cm×1.5 cm flaps were designed with symmetry axis of left lateral thoracic artery, thoracic dorsal artery and superficial abdominal artery. A model of ischemic flap was established by clamping pedicle with miniature artery clip. Basic blood flow of flap in each group were detected by full-field laser perfusion imager (FLPI), after 7 days reperfusion, percent necrosis of flap was measured. The optimal model of murine skin flap for ischemia/reperfusion was determined by statistical analysis.(2)In the experiment two, the remaining 72 male ICR mice were divided into 6 groups (n=12), ⅰ, ⅱ, ⅲ, ⅳ, ⅴ, ⅵ group according to the time of the vessel pedicled clamped(time of ischemia), the corresponding ischemia time respectively was 0, 1.5, 3.0, 5.0, 8.0, 10.0 h. Blood flow during ischemia and reperfusion were monitored and analysised using FLPI. After 7 days reperfusion, the survival of the flap was observed and the rate of skin flap necrosis was calculated. Flap tissues of half of the mice in each group were taken after 24.0 h of reperfusion. Histomorphological changes were analyzed by hematoxylin- eosin staining. Dichlorofluorescein diacetate probe method was used to detect the content of reactive oxygen species. Terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labing was used to detect apoptosis and calculate the positive index of apoptotic cells. The content of tumor necrosis factor-α(TNF- α)was determined by enzyme-linked immunosorbent assay (ELISA). Data were processed with one-way analysis of variance and t test.

In the experiment one, the results showed that the basal perfusion blood flow of the mice thoracic flap was (320.2±7.5) PU, the basal perfusion blood flow of dorsal skin flap was (156.3±12.4) PU, the basal perfusion blood flow of epigastric skin flap was (78.5±5.5) PU, and the differences was statistically significant (F=174.8, P<0.05). In the thoracic skin flap group, the necrosis rates of the flap were different in every ischemic time periods, the difference was statistically significant (F=261.7, P<0.05), meanwhile, the necrosis rates of the flap were different in every ischemic time periods in the dorsal skin flap group and in the epigastric flap skin group, the differences were statistically significant (F=39.6, 235.1; with P values below 0.05). In the experiment two, the results showed that the mice thoracic skin flap could tolerate ischemia within 1.5 hours, the flap reperfusion blood flow recovered fast. The necrosis rate of groupⅰ, ⅱ was (1.20±0.15)%, (6.50±0.24)%, which were less than 10.00%. The content of reactive oxygen species, the positive index of apoptotic cells, TNF-α in groupⅰwere 1.00±0.12, (3.2±0.1)%、(2.09±0.93) μg/μL, and in group ⅱ were 1.26±0.07, (4.3±0.1)%, (3.63±0.42) μg/μL, which were increased compared with groupⅰ, the differences were statistically significant(t=3.58, 6.77, 3.03; with P values below 0.05). In group ⅲ, ⅳ, the flap reperfusion blood flow recovered slowly, flap necrosis rate, relative multiples of reactive oxygen species, the positive index of apoptotic cells, TNF-α were(48.2±3.57)%, 2.34±0.17, (22.2±1.4)%, (4.79±0.72) μg/μL, which were increased significantly compared with group ⅱ, the differences were statistically significant(t=11.52, 10.50, 12.85, 2.80; with P values below 0.05). In groupⅴ, ⅵ, the flap reperfusion blood flow recovered very slowly. Hematoxylin - eosin staining showed tissue hyperemia and intravascular thrombosis. The necrosis rate and TNF-α were (75.7±3.30)%, (6.10±0.56)%, which were increased significantly compared with group ⅵ, the differences were statistically significant (t=6.59, 2.86; with P values below 0.05). Relative multiples of reactive oxygen species was 1.47±0.21 in groupⅴ, which was decreased significantly compared with group ⅵ, the difference was statistically significant(t=5.55, P=0.005); the positive index of apoptotic cells was (20.5±2.2)%, which was no significant difference compared with group ⅳ, the difference was statistically significant (t=0.15, P=0.88).

Compared with the model of mice dorsal skin flap and epigastric skin flap, the perfusion blood flow of mice thoracic skin flap model was higher and more stable, and the change of necrosis rate caused by different ischemic time was more statistically significant. Ischemia reperfusion can cause the injury of the thoracic skin flap in mice, with the prolongation of the ischemia time, reperfusion blood flow is more decreased, injury of the flap becomes worse. The mechanism of ischemia reperfusion injury to the skin flap may be that after ischemia reperfusion, a large amount of reactive oxygen species is produced, leading to vasoconstriction and affecting microcirculation. At the same time reactive oxygen species results in the accumulation of numerous neutrophils, local inflammatory reaction aggravation, leading to the aggravation of the flap injury.