大鼠烫伤创面感染模型的研制

Reproduction of a rat model of burn with infection

目的 拟建立稳定的大鼠烫伤创面感染模型,以便于进行相关防治研究.方法 （1）取50只SD大鼠,使用恒温恒压烫伤仪,以底面积4.5 cm2、质量0.5kg的80℃圆柱形烫头垂直接触大鼠脊柱左右两侧皮肤,致伤4、6、8、10、12 s（每种致伤时间10只大鼠,左右侧烫伤时间相同）制作烫伤模型.伤后24 h,观察创面大体情况,记录左侧创面愈合时间,取右侧创面组织行组织学观察,根据结果筛选浅Ⅱ度、深Ⅱ度创面致伤时间.（2）另取36只SD大鼠,按随机数字表法分为浅Ⅱ度组、深Ⅱ度组,每组18只,按照前述方法与选定的致伤时间分别制成浅Ⅱ度、深Ⅱ度烫伤创面.伤后即刻在2组大鼠一侧创面分别接种0.1 mL含1×10^9、1×10^7、1×10^5CFU铜绿假单胞菌标准菌株ATCC 27853的菌液（每种菌量6只大鼠）,在另一侧创面涂抹等体积生理盐水作为对照.接种细菌后24 h HE染色观察创面炎症反应情况;接种细菌后1、2、3、5、7、14 d进行革兰染色及生化反应鉴定菌种,检测并计算痂下细菌含量;记录2组大鼠创面愈合时间.对数据行t检验.结果 （1）根据大鼠创面愈合时间及组织学检查结果,筛选出烫伤6 s和8 s分别为浅Ⅱ度和深Ⅱ度创面的致伤时间.（2）浅Ⅱ度组大鼠仅接种1×10^9CFU细菌的创面有少许炎性细胞浸润;深Ⅱ度组接种1×10^9、1×10^7CFU细菌创面均有炎性细胞浸润,前者浸润更明显.（3）创面细菌鉴定结果为铜绿假单胞菌.浅Ⅱ度组创面接种各种菌量后14 d内,痂下细菌含量绝大多数低于1×10^5CFU/g;深Ⅱ度组创面接种1×10^9 CFU细菌后14 d内,痂下细菌含量均高于1×10^5CFU/g并呈持续上升趋势.（4）浅Ⅱ度组接种1×10^9、1×10^7、1×10^5 CFU细菌的创面与生理盐水对照创面愈合时间相近（t值分别为1.26、0.29、1.07,P值均大于0.05）;深Ⅱ度组接种1×10^9CFU细菌创面愈合时间[（22.5±1.0）d]较生理盐水对照创面[（19.4±1.6）d]明显延长（t=2.73,P＜0.05）.结论 大鼠深Ⅱ度烫伤创面接种1×10^9 CFU铜绿假单胞菌标准菌株ATCC 27853,可作为感染创面防治研究的实验模型,该模型稳定、重复性较高.

Objective To reproduce a reliable rat model of burn with infection for the study of prevention and treatment of infected wound. Methods （ 1 ） Electrical burn producing apparatus equipped with constant temperature （80 ℃ ） and pressure （0.5 kg） was used to reproduce burn injury （with area of 4.5 cm2 ） on both sides of the back in 50 SD rats for different duration （4, 6, 8, 10, 12 s） , with 10 rats for each burn duration. On post burn day （PBD） 1, gross condition of wounds was observed with naked eyes.Wounds on the left side were used to observe healing time. The wounds on the right side were used for histological observation to determine the depth of injury, and they were classified into superficial and deep partialthickness injury. （2） Another 36 SD rats were divided into A （inflicted with superficial partial-thickness burn, n = 18） and B （inflicted with deep partial-thickness burn, n = 18） groups according to the random number table. Rats in both groups were treated in accordance with method of preliminary experiment. Immediately after burn, 0. 1 mL of liquid containing 1×10^9, 1×10^7, 1×10^5 CFU Pseudomonas aeruginosa （PA） ATCC 27853 was respectively inoculated to the wounds on one side （with 6 rats for each amount） ,while the wounds on the other side were treated with the same volume of normal saline as control. Inflammatory reaction of wounds was examined with HE staining on post inoculation day （PID） 1. On PID 1, 2, 3,5, 7, and 14, the number of subeschar bacteria was respectively counted and the bacteria were identified with Gram stain and biochemical reaction. Wound healing time was recorded. Data were processed with t test. Results （1） Burn for 6, 8 s was respectively identified as injury time resulting in superficial or deep partial-thickness injury according to histological observation and wound healing time. （2） Obvious inflammatory cell infiltration was observed in the wounds in B group which were inoculated with 1×10^7 , 1 ×109 CFU PA, and the infiltration was less marked in A group with inoculation of 1×10^9 CFU PA. （3） The bacteria isolated from wounds of A and B groups was identified as PA. The subeschar bacteria count within PID 14 in A group, in which different amount of PA was inoculated, was mostly less than 1×10^5 CFU/g of tissue, while that in B group in which 1×10^9 CFU PA was inoculated was more than 1×10^5 CFU/g of tissue. （4） There was no obvious difference in wound healing time between wounds inoculated with different amount of PA and wounds treated with normal saline in A group （ with t value respectively 1.26, 0. 29, 1.07,P values all above 0.05 ）. Wound healing time of wounds in B group, in which 1×10^9 CFU PA was inoculated, was longer as compared with that treated with normal saline [（22.5 ± 1.0） d vs. （ 19.4 ± 1.6） d, t =2.73, P 〈0. 05]. Conclusions In rat, deep partial-thickness burn wound inoculated with 1×10^9 CFU PA ATCC 27853 is a reliable model with high reproducibility for the study of infection of burn wound.