Role and mechanism of infrared thermography in early identification of necrosis risk in random-pattern flaps
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摘要:
目的 探讨红外热成像（IRT）技术对随意型皮瓣坏死风险的早期识别作用及其机制。 方法 该研究为成组设计实验研究。于92只6~8周龄雄性C57BL/6小鼠背部建立随意型皮瓣模型,从尾部到头部划分为面积相等的Ⅰ、Ⅱ、Ⅲ区,用红外热像仪测量3个区术后0（即刻）、3、7 d的相对温度（目标区与对照区的温度差值）。术后3、7 d,通过受试者操作特征（ROC）曲线分析皮瓣Ⅱ区相对温度对皮瓣坏死的预测效能,并确定最佳截断值。根据术后3 d皮瓣Ⅱ区相对温度,将小鼠分为>-0.1 ℃组、≤-0.1 ℃组；根据术后7 d皮瓣Ⅱ区相对温度,将小鼠分为>0.1 ℃组、≤0.1 ℃组,每组各4只。计算前2组小鼠术后3 d和后2组小鼠术后7 d皮瓣Ⅱ区存活面积百分比,采用激光多普勒血流成像仪测量相应组别相应时间点皮瓣Ⅱ区血流灌注量。术后3 d,取>-0.1 ℃组、≤-0.1 ℃组小鼠皮瓣Ⅱ区组织,行转录组测序筛选差异表达基因（DEG）,行免疫荧光染色后计算诱导型一氧化氮合酶（iNOS）/CD68、精氨酸酶1（ARG1）/CD68比值,行免疫组织化学染色检测趋化因子配体2（CCL2）的表达,行二氢乙锭染色检测二氢乙锭的表达,行实时荧光定量反转录PCR法检测肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）和IL-10的mRNA表达,行蛋白质印迹法检测CD68、iNOS、ARG1、血红素氧合酶1（HO-1）及超氧化物歧化酶1（SOD1）的蛋白表达。 结果 术后0~7 d,皮瓣Ⅰ区和Ⅱ区相对温度呈升高趋势,而皮瓣Ⅲ区相对温度呈先升高后降低的趋势。术后3、7 d,皮瓣Ⅱ区相对温度预测皮瓣坏死的ROC曲线下面积分别为0.82、0.85（95%CI分别为0.72~0.93、0.78~0.93）,最佳截断值分别为-0.1、0.1 ℃。术后3 d,≤-0.1 ℃组与>-0.1 ℃组小鼠皮瓣Ⅱ区存活面积百分比及血流灌注量比较,差异均无统计学意义（P>0.05）；术后7 d,≤0.1 ℃组小鼠皮瓣Ⅱ区存活面积百分比显著低于>0.1 ℃组（t=4.19,P<0.05）,血流灌注量显著少于>0.1 ℃组（t=5.58,P<0.05）。术后3 d,相较于>-0.1 ℃组,≤-0.1 ℃组小鼠皮瓣Ⅱ区组织中存在2 849个DEG,其中1 509个上调DEG、1 340个下调DEG。术后3 d,>-0.1 ℃组小鼠皮瓣Ⅱ区组织中iNOS/CD68比值显著低于≤-0.1 ℃组（t=4.97,P<0.05）,ARG1/CD68比值显著高于≤-0.1 ℃组（t=3.42,P<0.05）,CCL2、二氢乙锭的表达和TNF-α、IL-1β的mRNA表达及CD68、iNOS的蛋白表达均显著低于≤-0.1 ℃组（t值分别为4.82、4.51、4.43、5.47、-3.69、-6.14,P<0.05）,IL-10的mRNA表达及ARG1、SOD1与HO-1的蛋白表达均显著高于≤-0.1 ℃组（t值分别为8.58、8.61、6.24、11.03,P<0.05）。 结论 IRT技术可通过监测皮瓣温度实现小鼠随意型皮瓣坏死风险的早期客观预测。术后3 d皮瓣Ⅱ区相对温度≤-0.1 ℃时,组织呈现显著炎症反应、免疫细胞浸润及氧化应激状态。CCL2可能是诱导巨噬细胞极化的关键趋化因子。 Abstract:Objective To investigate the role and mechanism of infrared thermography (IRT) in early identification of necrosis risk in random-pattern flaps (RPFs). Methods This study was designed as a group-comparison experimental study. A RPF model was established on the dorsum of 92 male C57BL/6 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) of the three zones were measured using an infrared thermal imager at postoperative day (PPD) 0 (immediately), 3, and 7. At PPD 3 and 7, receiver operating characteristic (ROC) curves were used to evaluate the predictive efficacy of the RT of zone Ⅱ for flap necrosis, and the optimal cutoff values were determined. Based on the RT of flap zone Ⅱ at PPD 3, mice were divided into >-0.1 ℃ group and ≤-0.1 ℃ group; based on the RT of flap zone Ⅱ at PPD 7, mice were divided into >0.1 ℃ group and ≤0.1 ℃ group, with 4 mice in each group. The percentage of surviving area of flap zone Ⅱ was calculated for the first two groups of mice at PPD 3 and for the latter two groups of mice at PPD 7. Laser Doppler perfusion imaging was used to measure blood perfusion volume in flap zone Ⅱ of the above groups at the corresponding time points. At PPD 3, the tissue from flap zone Ⅱ of >-0.1 ℃ and ≤-0.1 ℃ groups of mice was collected for transcriptomic sequencing to identify differentially expressed genes (DEGs), 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 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 mRNA expressions of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-10, and for Western blotting to detect protein expressions of CD68, iNOS, ARG1, heme oxygenase-1 (HO-1), and superoxide dismutase 1 (SOD1). Results From PPD 0 to 7, RTs in flap zones Ⅰ and Ⅱ showed an increasing trend, while RTs in flap zone Ⅲ showed a trend of initial increase followed by decrease. At PPD 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 (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 PPD 3, there were no statistically significant differences in the percentage of surviving area or blood perfusion volume in flap zone Ⅱ between ≤-0.1 ℃ group and >-0.1 ℃ group of mice (P>0.05). At PPD 7, the percentage of surviving area (t=4.19, P<0.05) and blood perfusion volume (t=5.58, P<0.05) in flap zone Ⅱ were significantly decreased in ≤0.1 ℃ group of mice compared with those in >0.1 ℃ group. At PPD 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 PPD 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 PPD 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. -
参考文献
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图 3 各组小鼠术后不同时间点随意型皮瓣存活情况及血流灌注情况。3A、3B.分别为>-0.1 ℃组和≤-0.1 ℃组术后3 d皮瓣存活情况,图3A和图3B皮瓣Ⅱ区存活面积相近；3C、3D.分别为>0.1 ℃组和≤0.1 ℃组术后7 d皮瓣存活情况,图3C皮瓣Ⅱ区存活面积大于图3D；3E、3F.分别为>-0.1 ℃组和≤-0.1 ℃组术后3 d皮瓣血流灌注情况,图3E和图3F皮瓣Ⅱ区血流灌注量相近；3G、3H.分别为>0.1 ℃组和≤0.1 ℃组术后7 d皮瓣血流灌注情况,图3G皮瓣Ⅱ区血流灌注量多于图3H
图 4 2组小鼠术后7 d随意型皮瓣Ⅱ区组织中CHP荧光强度及CD34和α-SMA双阳性细胞数。4A、4B.分别为>0.1 ℃组和≤0.1 ℃组CHP荧光强度 荧光显微镜 ×200,图4A中CHP荧光强度低于图4B；4C、4D.分别为>0.1 ℃组和≤0.1 ℃组CD34和α-SMA双阳性细胞数 Alexa Fluor 488-Alexa Fluor 594-4',6-二脒基-2-苯基吲哚 ×200,图4C中CD34和α-SMA双阳性细胞数多于图4D
注：胶原蛋白杂交肽（CHP）阳性染色为绿色,CD34和α-平滑肌肌动蛋白（α-SMA）阳性染色分别为红色、绿色
图 5 2组小鼠术后3 d随意型皮瓣Ⅱ区组织中巨噬细胞极化、炎症趋化及氧化应激情况。5A~5D和5E~5H.分别为>-0.1 ℃组和≤-0.1 ℃组,图5A、5E和5B、5F分别为M1型（iNOS/CD68比值）、M2型（ARG1/CD68比值）巨噬细胞情况 Alexa Fluor 488-Alexa Fluor 594-4',6-二脒基-2-苯基吲哚 ×200,图5C和5G为CCL2表达情况 二氨基联苯胺-苏木精 ×200,图5D和5H为二氢乙锭表达情况 二氢乙锭-4',6-二脒基-2-苯基吲哚 ×200；与>-0.1℃组比较,≤-0.1 ℃组皮瓣Ⅱ区组织中iNOS/CD68比值升高、ARG1/CD68比值降低、CCL2和二氢乙锭的表达升高
注：CD68阳性染色为绿色,诱导型一氧化氮合酶（iNOS）、精氨酸酶1（ARG1）阳性染色为红色；趋化因子配体2（CCL2）阳性染色为棕色,二氢乙锭阳性染色为红色
图 6 蛋白质印迹法检测的2组小鼠术后3 d皮瓣Ⅱ区组织中巨噬细胞相关蛋白及抗氧化酶的蛋白表达
注：iNOS为诱导型一氧化氮合酶,ARG1为精氨酸酶1,SOD1为超氧化物歧化酶1,HO-1为血红素氧合酶1,GAPDH为3-磷酸甘油醛脱氢酶；条带上方1~3为>-0.1 ℃组的3个样本,4~6为≤-0.1 ℃组的3个样本
Table 1. 小鼠术后各时间点随意型皮瓣各区的相对温度（℃,
区域 鼠数（只） 术后0 d（即刻） 术后3 d 术后7 d Ⅰ区 92 -1.71±1.03 -0.71±0.52 -0.44±0.43 Ⅱ区 92 -0.85±0.63 -0.14±0.37 0.03±0.33 Ⅲ区 92 -0.40±0.46 -0.01±0.32 -0.13±0.30 F值 133.20 162.00 77.17 P值 <0.001 <0.001 <0.001 注：相对温度指皮瓣各区温度与对照区温度的差值 
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