严重烫伤大鼠淋巴循环中三种重要炎症介质的变化

目的 了解淋巴途径在烫伤大鼠肠道细菌移位中的作用.方法 制备羰花青荧光染料CM-DIL标记的大肠埃希菌菌液(数量级109CFU/L).将60只成年雄性Wistar大鼠按照信封法随机分为烫伤组、假伤组,每组30只.2组大鼠经胃灌注已制备的标记菌液0.5 mL后,烫伤组致30%TBSA深Ⅱ度烫伤,伤后立即行液体复苏;假伤组大鼠皮肤经25℃水浴10 s模拟烫伤并同法复苏.采集2组大鼠伤后2、24、72 h(每时相点10只)的肠系膜淋巴结(MLN)、肝脏、肠系膜淋巴液(MLF)、肝静脉血标本.分别采用荧光示踪法和细菌培养法检测细菌移位情况;用鲎试剂显色基质法定量测定上述4种标本中内毒素含量,并计算MLF和肝静脉血的内毒素携带量.对实验数据行t检验或单因素方差分析.结果 (1)荧光示踪法检测显示活菌呈短棒状,液体标本中可见单体或两三个联体的可移动活菌;死菌呈不规则碎片状.假伤组伤后2 h有少量标记菌,24 h数量达高峰;烫伤组伤后2 h标记菌较多,24、72 h仍处于较高水平.烫伤组各种标本比较,MLN内标记菌最多,伤后24 h达高峰[(5872±1976)×103 CFU/g],明显多于假伤组[(216±110)×103 CFU/g,t=30.129,P=0.000],72 h有所减少但仍多于假伤组(t=4.323,P=0.000);血液内标记菌最少.(2)细菌培养法检测:假伤组120份标本中,29份细菌培养呈阳性占24.2%;烫伤组120份标本中,72份培养阳性占60.0%.2组大鼠除血液标本在各时相点均未检出活菌外,其余3种标本伤后2 h或24 h可培养出活菌.烫伤组MLN和肝脏较其余2种组织检出更多活菌;伤后24 h,该组MLN、肝脏以及MLF标本中细菌数量均明显多于假伤组(t值分别为4.353、4.354、4.965,P值均等于0.000).(3)内毒素含量:烫伤组大鼠伤后各时相点4种标本的内毒素含量均高于假伤组,其中肝脏、MLF内毒素含量伤后2 h即达高峰.此时相点烫伤组4种组织间内毒素含量比较,差异有统计学意义(F=258.47,P=0.000),其中肝脏、MLN、MLF内毒素含量较高,且明显高于假伤组(t值分别为43.378、43.123、22.423,P值均等于0.000);MLF内毒素含量约为血液中含量的9倍.(4)烫伤组伤后各时相点MLF、血液内毒素携带量均高于假伤组.结论 用CM-DIL标记细菌能够较全面反映细菌移位情况,淋巴途径在细菌移位中发挥重要作用.

Abstract：

Objective To investigate the role of lymphatics in bacterial translocation from intestine of rats with burn. Methods Escherichia coli (E. coli) labeled with chloromethylbenzamidodialkylcarbocyanine (CM-DIL) were prepared. Sixty adult male Wistar rats were randomly divided into scald group and sham injury group according to the envelope method, with 30 rats in each group. Rats in both groups were gavaged with 0. 5 mL fluid containing CM-DIL-labeled E. coli. Rats in scald group were inflicted with 30% TBSA deep partial-thickness scald (verified by pathological section) and resuscitated with fluid. Rats in sham injury group were sham injured by bathing in 25 ℃ water for 10 s(verified by pathological section)and also received with fluid infusion. Mesenteric lymph node (MLN), liver, mesenteric lymph fluid (MLF), and liver vein blood (LVB) were harvested at post injury hour (PIH) 2, 24, and 72. Bacteria translocation was detected with fluorescent tracing technique and bacteria culture. The endotoxin content in above-mentioned four kinds of specimens was quantitatively determined with chromogenic substrate limulus amebocyte lysate. The carrying capacity of endotoxin in MLF and LVB was calculated. Data were processed with t test or one-way analysis of variance. Results (1) Living bacteria were in short-stick form, and they were seen moving in single or in doubles or triples in sample fluid. Dead bacteria were in irregular aggregates. Labeled bacteria in small amount were detected in sham injury group, their number peaked at PIH 24. A large amount of labeled bacteria were detected in scald group at PIH 2, which peaked at PIH 24 and decreased at PIH 72. The largest amount of labeled bacteria were found in MLN in scald group as compared to those in the other samples, and the number peaked at PIH 24 [(5872 ± 1976) × 103 CFU/g], which was obviously higher than that [(216 ± 110) × 103 CFU/g, t =30. 129, P =0.000] in sham injury group. The number of bacteria decreased at PIH 72, but it was still significantly different from that in sham injury group ( t =4. 323, P =0.000). The number of bacteria in LVB was the smallest. (2) 29 (24.2%) samples out of the 120 samples in sham injury group were positive for bacteria. 72 (60.0%) samples out of the 120 samples in scald group were positive for bacteria. No alive bacterium was detected at any time point in LVB sample in both group; the other three samples were detected with alive bacteria since PIH 2. There were more alive bacteria detected in MLN and liver as compared with the other two kinds of samples in scald group. The amount of bacteria in MLN, liver, and MLF in scald group were higher than those in sham injury group(with t value respectively 4. 353, 4. 354, 4. 965, P values all equal to 0. 000). (3) The endotoxin level in each kind of sample at each time point was obviously higher in scald group than that in sham injury group, and it peaked at PIH 2 in liver and MLF. The difference of endotoxin level among 4 kinds of samples in scald group at PIH 2 was statistically significant(F = 258.47, P = 0. 000) , and the endotoxin level was higher in liver, MLN, and MLF. They were obviously higher than those in sham injury group(with t value respectively 43. 378, 43. 123, 22. 423, P values all equal to 0. 000). The endotoxin level in MLF was 9 times of that in LVB. (4) The carrying capacity of endotoxin in LVB and MLF at each time point in scald group was higher than that in sham injury group. Conclusions CM-DIL marked bacteria can reflect the microbial translocation condition. The lymphatic route is an important pathway for bacteria translocation.

Role of lymphatics in bacterial translocation from intestine in burn rats

Objective To investigate the role of lymphatics in bacterial translocation from intestine of rats with burn. Methods Escherichia coli (E. coli) labeled with chloromethylbenzamidodialkylcarbocyanine (CM-DIL) were prepared. Sixty adult male Wistar rats were randomly divided into scald group and sham injury group according to the envelope method, with 30 rats in each group. Rats in both groups were gavaged with 0. 5 mL fluid containing CM-DIL-labeled E. coli. Rats in scald group were inflicted with 30% TBSA deep partial-thickness scald (verified by pathological section) and resuscitated with fluid. Rats in sham injury group were sham injured by bathing in 25 ℃ water for 10 s(verified by pathological section)and also received with fluid infusion. Mesenteric lymph node (MLN), liver, mesenteric lymph fluid (MLF), and liver vein blood (LVB) were harvested at post injury hour (PIH) 2, 24, and 72. Bacteria translocation was detected with fluorescent tracing technique and bacteria culture. The endotoxin content in above-mentioned four kinds of specimens was quantitatively determined with chromogenic substrate limulus amebocyte lysate. The carrying capacity of endotoxin in MLF and LVB was calculated. Data were processed with t test or one-way analysis of variance. Results (1) Living bacteria were in short-stick form, and they were seen moving in single or in doubles or triples in sample fluid. Dead bacteria were in irregular aggregates. Labeled bacteria in small amount were detected in sham injury group, their number peaked at PIH 24. A large amount of labeled bacteria were detected in scald group at PIH 2, which peaked at PIH 24 and decreased at PIH 72. The largest amount of labeled bacteria were found in MLN in scald group as compared to those in the other samples, and the number peaked at PIH 24 [(5872 ± 1976) × 103 CFU/g], which was obviously higher than that [(216 ± 110) × 103 CFU/g, t =30. 129, P =0.000] in sham injury group. The number of bacteria decreased at PIH 72, but it was still significantly different from that in sham injury group ( t =4. 323, P =0.000). The number of bacteria in LVB was the smallest. (2) 29 (24.2%) samples out of the 120 samples in sham injury group were positive for bacteria. 72 (60.0%) samples out of the 120 samples in scald group were positive for bacteria. No alive bacterium was detected at any time point in LVB sample in both group; the other three samples were detected with alive bacteria since PIH 2. There were more alive bacteria detected in MLN and liver as compared with the other two kinds of samples in scald group. The amount of bacteria in MLN, liver, and MLF in scald group were higher than those in sham injury group(with t value respectively 4. 353, 4. 354, 4. 965, P values all equal to 0. 000). (3) The endotoxin level in each kind of sample at each time point was obviously higher in scald group than that in sham injury group, and it peaked at PIH 2 in liver and MLF. The difference of endotoxin level among 4 kinds of samples in scald group at PIH 2 was statistically significant(F = 258.47, P = 0. 000) , and the endotoxin level was higher in liver, MLN, and MLF. They were obviously higher than those in sham injury group(with t value respectively 43. 378, 43. 123, 22. 423, P values all equal to 0. 000). The endotoxin level in MLF was 9 times of that in LVB. (4) The carrying capacity of endotoxin in LVB and MLF at each time point in scald group was higher than that in sham injury group. Conclusions CM-DIL marked bacteria can reflect the microbial translocation condition. The lymphatic route is an important pathway for bacteria translocation.