Hematoxylin and Eosin Staining
At the 7th POD, six tissue specimens from the flap II region (central tissue) measuring 1cm × 1cm were collected after the rats were sacrificed with an excess sodium pentobarbital dose. After 24 hours of fixation in 4% (v/m) paraformaldehyde, these specimens were embedded in paraffin wax and transversely sectioned (4 mm thickness). We randomly selected the number of blood vessel cross-sections per unit area in different fields of view (/mm2) under the microscope (Olympus Corp, Japan) to compute the mean blood vessel density of the flap tissue in order to evaluate the microcirculation level.