[Role and mechanism of infrared thermography in early identification of necrosis risk in random-pattern flaps of mice].

Abstract

Objective: To investigate the role and mechanism of infrared thermography (IRT) in early identification of necrosis risk in random-pattern flaps (RPFs) of mice. Methods: This study was an experimental study with a grouped design. A RPF model was established on the dorsum of 92 male C57BL/6J mice aged 6-8 weeks. The flap was divided into three equal zones (zones Ⅰ, Ⅱ, and Ⅲ) from the caudal to the cephalic end. The relative temperatures (RTs, the difference between the target zones and the normal control zone) of the three zones were measured using an infrared thermal imager at postoperative day (POD) 0 (immediately), 3, and 7. At POD 3 and 7, receiver operating characteristic (ROC) curves were used to evaluate the predictive efficacy of the RT of flap zone Ⅱ for flap necrosis and to determine the optimal cutoff values. Based on the RT of flap zone Ⅱ at POD 3, mice were divided into >-0.1 ℃ group and ≤-0.1 ℃ group; based on the RT of flap zone Ⅱ at POD 7, mice were divided into >0.1 ℃ group and ≤0.1 ℃ group. The survival rate of flaps was calculated for the first two groups of mice at POD 3 and for the latter two groups of mice at POD 7. Laser Doppler perfusion imaging was used to measure blood perfusion volume of flaps in the above groups at the corresponding time points. At POD 3, the tissue of flap zone Ⅱ from >-0.1 ℃ and ≤-0.1 ℃ groups of mice was collected for transcriptomic sequencing to screen for differentially expressed genes (DEGs) with significantly differential expression, for immunofluorescence staining to calculate the ratios of inducible nitric oxide synthase (iNOS)/CD68 and arginase 1 (ARG1)/CD68, for immunohistochemical staining to detect the expression of C-C motif chemokine ligand 2 (CCL2), for dihydroethidium (DHE) staining to detect the expression of DHE, for real-time fluorescence quantitative reverse transcription polymerase chain reaction to measure the mRNA expressions of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-10, and for Western blotting to detect the protein expressions of CD68, iNOS, ARG1, heme oxygenase-1 (HO-1), and superoxide dismutase 1 (SOD1). The sample size was three for the DEGs screening and protein expression detecting experiments, and 4 for the remaining experiments. Results: From POD 0 to 7, RTs of flap zones Ⅰ and Ⅱ showed an increasing trend, while RTs in flap zone Ⅲ rose initially and then decreased. At POD 3 and 7, the areas under the ROC curves of the RT of flap zone Ⅱ for predicting flap necrosis were 0.82 and 0.85, respectively (with 95% CIs of 0.72-0.93 and 0.78-0.93, respectively), with optimal cutoff values of -0.1 and 0.1 ℃, respectively. At POD 3, there were no statistically significant differences in the survival rate or blood perfusion volume of flaps between ≤-0.1 ℃ group and >-0.1 ℃ group of mice (P>0.05). At POD 7, the survival rate (t=4.19, P<0.05) and blood perfusion volume (t=5.58, P<0.05) of flaps were significantly decreased in ≤0.1 ℃ group of mice compared with those in >0.1 ℃ group. At POD 3, compared with that in >-0.1 ℃ group, there were 2 849 DEGs in tissue of flap zone Ⅱ in ≤-0.1 ℃ group of mice, including 1 509 upregulated DEGs and 1 340 downregulated DEGs. At POD 3, there were significantly lower iNOS/CD68 ratio (t=4.97, P<0.05) and significantly higher ARG1/CD68 ratio (t=3.42, P<0.05), significantly lower expressions of CCL2 and DHE, mRNA expressions of TNF-α and IL-1β, as well as protein expressions of CD68 and iNOS (with t values of 4.82, 4.51, 4.43, 5.47, 3.69, and 6.14, respectively, P<0.05), and significantly higher mRNA expression of IL-10 and protein expressions of ARG1, SOD1, and HO-1 (with t values of 8.58, 8.61, 6.24, and 11.03, respectively, P<0.05) in tissue of flap zone Ⅱ in >-0.1 ℃ group of mice compared with those in ≤-0.1 ℃ group. Conclusions: IRT enables early and objective prediction of necrosis risk of RPFs in mice through monitoring flap temperature. The RT ≤-0.1 ℃ in flap zone Ⅱ at POD 3 is associated with marked inflammatory response, immune cell infiltration, and oxidative stress in the tissue. CCL2 may serve as a key chemokine inducing macrophage polarization.