Establishment and efficacy evaluation of a rabbit lower limb partial-thickness scald model
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摘要:
目的 建立一种兔下肢Ⅱ度烫伤模型并评价其效果。 方法 该研究为重复测量设计和成组设计实验研究。取40只6~8月龄健康雄性新西兰兔,按照随机数字表法分为烫伤组（30只）和正常对照组（10只）。对烫伤组兔于麻醉后采用桶状恒温水浴锅和自制兔烫伤固定支架于水温70 ℃中浸烫右下肢12 s制作兔下肢Ⅱ度烫伤模型, 伤后6 h内补液。对正常对照组仅与烫伤组在同一时间点行脱毛和麻醉,不行后续烫伤、补液处理。任意选取10只烫伤组兔,分别于伤前及伤后6、24 h,行苏木精-伊红染色后观察创面组织形态。另取10只烫伤组兔,分别于伤前及伤后0（即刻）、1、3、6、12、24、48 h,使用激光多普勒血流仪观测右下肢皮肤血流灌注量。取烫伤组剩余10只兔,分别于伤前及伤后10 s、20 s、40 s、2 min、10 min、20 min、40 min、1 h、2 h、3 h、4 h、5 h、6 h,采用非接触的红外温度传感器和接触式温度传感器测量右下肢皮下和深部肌肉温度。取行血流灌注量观测后的10只烫伤组兔,分别于伤前及伤后1、2、3、4、5、6、24、48、72 h,测量右下肢周径；记录足背部、足底部及小腿部创面愈合时间。取正常对照组及行温度测量后的烫伤组各10只兔,采用酶联免疫吸附测定法检测血清中白细胞介素-1β（IL-1β）、IL-6、肿瘤坏死因子-α（TNF-α）、超氧化物歧化酶（SOD）、丙二醛水平。 结果 烫伤组兔伤前右下肢表皮、皮肤附属器完整；伤后6 h,可见表皮连续性中断,部分皮肤附属器消失,组织间隙增加,水肿增加；伤后24 h,皮肤附属器萎缩消失,组织水肿明显增加,真皮浅层炎症细胞浸润增加。伤前及伤后0、1、3、6、12、24、48 h,烫伤组兔右下肢皮肤血流灌注量分别为（1.00±0.26）、（1.03±0.29）、（1.04±0.29）、（1.19±0.37）、（1.30±0.50）、（1.36±0.99）、（1.39±0.22）、（0.72±0.21）灌注单位（时间因素主效应,F=12.55,P<0.05）。烫伤组兔伤前及伤后10 s、20 s、2 min、10 min、20 min、40 min、1 h、2 h、3 h、4 h、5 h、6 h右下肢皮下温度均明显低于伤后40 s（P<0.05）,伤前及伤后10 s、20 s、40 s、2 min、20 min、40 min、1 h、2 h、3 h、4 h、5 h、6 h右下肢深部肌肉温度均明显低于伤后10 min（P<0.05）。烫伤组兔伤后24 h右下肢周径明显大于伤前及伤后1、2、3、4、5、6、48、72 h（P值均<0.05）。烫伤组兔足底部创面愈合时间明显短于足背部和小腿部（P值均<0.05）。伤后24 h,与正常对照组比较,烫伤组兔血清中IL-1β、IL-6、TNF-α、丙二醛水平均明显升高（t值分别为21.92、7.48、12.58、117.34,P<0.05）,SOD水平明显降低（t=117.34,P<0.05）。 结论 本研究建立的兔下肢Ⅱ度烫伤模型能够安全、稳定地模拟人类肢体烫伤后病理生理过程及特点,是研究人肢体Ⅱ度烫伤的较佳较好模型。 Abstract:Objective To establish a rabbit lower limb partial-thickness scald model and evaluate its efficacy. Methods This study was a repeated measures designed and group designed experimental study. Forty healthy male New Zealand rabbits aged 6 to 8 months were selected, and divided into scald group (30 rabbits) and normal control group (10 rabbits) according to the random number table method. The rabbits in scald group were anesthetized, then a bucket-type constant temperature water bath pot and a self-made rabbit scald fixation bracket were used to immerse the right lower limb in water at 70 ℃ for 12 s to prepare a rabbit lower limb partial-thickness scald model, then fluid supplementation were conducted within 6 h after injury. The rabbits in normal control group were subjected to hair removal and anesthesia as scald group at the same time point except for scald and fluid supplementation. Ten rabbits were randomly selected from scald group, and the wound tissue morphology was observed after hematoxylin-eosin staining before injury and at 6 and 24 h after injury, respectively. Another 10 rabbits in scald group were observed for skin blood perfusion in right lower limb using laser Doppler flowmetry before injury and at 0 (immediate), 1, 3, 6, 12, 24, and 48 h after injury, respectively. The remaining 10 rabbits in scald group were measured for subcutaneous and deep muscle temperatures in right lower limb using non-contact infrared temperature sensors and contact temperature sensors before injury and at 10 s, 20 s, 40 s, 2 min, 10 min, 20 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h after injury, respectively. After blood perfusion observation, the 10 rabbits in scald group were measured for right lower limb circumference before injury and at 1, 2, 3, 4, 5, 6, 24, 48, and 72 h after injury, respectively, and wound healing time of dorsal foot, plantar foot, and crural regions were recorded. Ten rabbits from normal control group and 10 rabbits after temperature measurement from scald group were taken to detect the serum levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD), and malondialdehyde by enzyme-linked immunosorbent assay. Results Before injury, epidermis and skin appendages of the right lower limb of rabbits in scald group were intact. At 6 h after injury, epidermal continuity was interrupted, partial skin appendages disappeared, interstitial spaces increased, and edema increased. At 24 h after injury, skin appendages atrophied and disappeared, tissue edema increased significantly, and inflammatory cell infiltration increased in superficial dermis. The skin blood perfusion of right lower limb of rabbits in scald group before injury and at 0, 1, 3, 6, 12, 24, and 48 h after injury was (1.00±0.26), (1.03±0.29), (1.04±0.29), (1.19±0.37), (1.30±0.50), (1.36±0.99), (1.39±0.22), and (0.72±0.21) perfusion units, respectively (main effect of time factor, F=12.55, P<0.05). The subcutaneous temperature in right lower limb of rabbits in scald group before injury and at 10 s, 20 s, 2 min, 10 min, 20 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h after injury was significantly lower than that at 40 s after injury (P<0.05). The deep muscle temperature in right lower limb of rabbits in scald group before injury and at 10 s, 20 s, 40 s, 2 min, 20 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h after injury was significantly lower than that at 10 min after injury (P<0.05). The right lower limb circumference in rabbits in scald group at 24 h after injury was significantly larger than that before injury and at 1, 2, 3, 4, 5, 6, 48, and 72 h after injury (with P values all <0.05). The wound healing time of plantar foot of rabbits in scald group was significantly shorter than that of dorsal foot and crural region (with P values both <0.05). At 24 h after injury, compared with those in normal control group, serum levels of IL-1β, IL-6, TNF-α, and malondialdehyde in rabbits in scald group were significantly increased (with t values of 21.92, 7.48, 12.58, and 117.34, respectively, P<0.05), while serum level of SOD was significantly decreased (t=117.34, P<0.05). Conclusions The rabbit lower limb partial-thickness scald model established in this study can safely and stably simulate the pathophysiological process and clinical characteristics of human limb scald, thus is a favorable model for studying partial-thickness scald of human limbs. -
Key words:
- Burns /
- Models, animal /
- Oxidative stress /
- Inflammation /
- Wound repair
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参考文献
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图 2 烫伤组兔各时间点右下肢大体形态与组织学变化。2A、2B、2C.分别为伤前、伤后6 h及伤后24 h右下肢大体形态,其中图2B较图2A皮肤发红伴红白相间,指蹼足背部皮肤肿胀,图2C较图2B肢体肿胀更加明显,足背及腿部皮肤出现类似Ⅱ度烫伤水泡结构；2D、2E、2F.分别为伤前、伤后6 h及伤后24 h右下肢烫伤组织损伤情况,其中图2E较图2D表皮连续性中断,毛囊等皮肤附属器部分受损后消失,图2F较图2E真皮间隙增加,组织水肿程度增加,炎症细胞浸润增加 苏木精-伊红 ×20
注：烫伤组兔右下肢致Ⅱ度烫伤
图 3 烫伤组兔各时间点右下肢皮肤血流灌注变化。3A、3B、3C、3D、3E、3F、3G、3H.分别为伤前及伤后0（即刻）、1、3、6、12、24、48 h激光多普勒动态扫描图,图3B血流灌注量明显多于图3A,随时间推移血流灌注增加,图3G血流灌注量最多,图3H比图3G血流灌注量少
注：烫伤组兔右下肢致Ⅱ度烫伤；黑色表示无血流灌注,绿色表示血流灌注少,红色表示血流灌注充足
Table 1. 烫伤组兔各时间点右下肢皮下和深部肌肉温度变化（℃,
时间点 样本数 皮下温度 深部肌肉温度 P1值 P2值 伤前 10 19.7±0.4 21.3±0.7 <0.001 <0.001 伤后10 s 10 31.3±0.5 26.2±1.2 <0.001 <0.001 伤后20 s 10 44.2±1.1 28.3±0.9 <0.001 <0.001 伤后40 s 10 60.2±0.5 30.3±1.5 — <0.001 伤后2 min 10 55.7±2.1 35.3±1.3 <0.001 <0.001 伤后10 min 10 50.7±1.5 39.7±0.4 <0.001 — 伤后20 min 10 44.3±1.2 37.3±1.2 <0.001 <0.001 伤后40 min 10 39.4±1.1 36.2±1.0 <0.001 <0.001 伤后1 h 10 34.3±1.3 34.7±0.9 <0.001 <0.001 伤后2 h 10 30.1±1.2 32.1±1.1 <0.001 <0.001 伤后3 h 10 29.6±0.5 29.7±1.2 <0.001 <0.001 伤后4 h 10 28.4±0.7 28.7±1.5 <0.001 <0.001 伤后5 h 10 28.1±1.1 28.1±1.1 <0.001 <0.001 伤后6 h 10 28.0±0.9 27.9±0.8 <0.001 <0.001 注：烫伤组兔右下肢致Ⅱ度烫伤；皮下温度中时间主效应,F=651.42,P<0.001；深部肌肉温度中时间主效应,F=271.63,P<0.001；P1值为伤后40 s与其他时间点皮下温度比较所得；P2值为伤后10 min与其他时间点深部肌肉温度比较所得；“—”表示无此项 
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Table 2. 2组兔伤后24 h血清中炎症因子和氧化应激因子水平比较（
组别 样本数 IL-1β（pg/mL） IL-6（pg/mL） TNF-α（pg/mL） SOD（U/mL） 丙二醛（nmol/mL） 正常对照组 10 15.1±0.3 123±15 112±11 150±23 210±11 烫伤组 10 28.6±1.9 194±26 217±24 90±17 930±16 t值 21.92 7.48 12.58 6.63 117.34 P值 <0.001 <0.001 <0.001 <0.001 <0.001 注：正常对照组兔不致伤,烫伤组兔致Ⅱ度烫伤；IL为白细胞介素,TNF-α为肿瘤坏死因子-α,SOD为超氧化物歧化酶
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