| Objective: Burn is a strong stress to the body, among which enteric mucus injury is the most severe and rapid, and then bacteria translocation will happen. Bacteria translocation, defined as the passage of both viable and nonviable microbes and microbial products, including endotoxin, across an anatomically intact intestinal barrier, has become a focus topic of surgical research. This process could be, at least in part, a predisposing factor for systemic infections, postinjury such as burns, multiple organ failure in the absence of a defined focus of infection. Reticuloendothelial system will be activated, when they deal with translocated endotoxin and bacteria, and release cytokine followed by cascade reactions. The organs will suffer a lot from too high level of cytokines.Previous studies revealed that translocation as measured by recovery of viable organisms from regional lymph nodes, liver, blood, thoracic duct lymph, increased by agents causing direct injury of mucosal cells and by indirect injury, which lack direct data of mesenteric lymph.In this study to elucidate the status of lymphatic route we developed an practical experimental animal model of lymphatic duct fistulation, compared the level of endotoxin, bacteria, cytokine and immune status in mesenteric lymph or mesenteric lymph nods with blood or liver.Methods: Adult male Wistar rats weighing 220 to 270 g were used for all experiment. Catheterization of the thoracic duct and mesenteric duct with median abdominal incision or oblique incision is performed with microsurgery technique and the lymph were drained for sampling. The caval vein is canulated, and the tip of the canula directed at the hepatic vein. All the animals were divided into 2 groups randomly: scalds group and sham group. MLNs, blood, mesenteric lymph and liver were harvested at 2h, 24h, 72h after burns.Escherichia coli were labeled with CM-Dil and lavaged, the translocated bacteria were determined with fluorometric method and tissue culture. Colony-forming units (CFU) were counted and the concentration of bacteria was calculated per gram of wet tissue, and the concentration of LPS (EU/ml) was then measured by the Limulus amoebocyte lysate assay.Lymph, plasma and the homogenate of MLNs or livers were diluted 1:10 and the IL-18, IL-10 concentration was determined by enzyme-linked immunosorbent assay (ELISA) techniques according to the manufacturer's instructions. The expression of HMGB-1 was determined via western blotting .The apoptosis of MLNs were assessed with electric microscope and flow cytometry with FITC-Annexin V/PI stain. Meanwhile, the change of T cell subsets of MLNs was get from flow cytometry.One more group received dermabrasion to the wound with dermabrader during shock stage.Results: Successful catheterization accounted 82.5% of all the experiment animals with microsurgery technique. The operation duration of mesenteric lymphatic duct and thoracic duct were 25.4±6.7min/ 29.2±7.4min.The difference of operation duration between oblique incision and median abdominal incision is not significant to mesenteric lymphatic catheterization. The average output of mesenteric lymph was close to each other.2. Bacteria labeled were found in all of the tissues. The amount of translocated organism began to increase at 2 hours after burn, and came to climax at 24 hours after burn, and then the translocated organism decreased at 72 hours after burn, while the proportion of the survival bacteria increased slightly. The concentration of LPS increased rapidly and the peak point is at 2 hours after burns. The concentration of LPS in lymph is ten times higher than that of blood, while the amount of LPS transported by lymph is much lesser than that of blood.3.The levels of cytokine: the level of IL-18 increased significantly at 2 hours after burn, came to peak level at 24 hours after burns and sustained at a relatively high level with 72 hours after burn. The concentration of IL-18 in MLNs was much higher than livers, whose level, however decreasing slightly with 72 hrs after burn. The level of 1L-10 did not increase significantly until 24hrs after burn. There was no HMGB1 protein determined at 2 hours after burns. The protein was expressed highly at 24 hours after burns and kept at a high level with 72 hrs.The apoptosis of MLNs: the apoptosis cell proportion of sham group was 8.6±0.57%, the apoptosis cell proportion of burn group was 16.5±0.75%,23.0±0.57 % , 72 27.5±1.02% at 2,24,72hrs respectively. The apoptosis cell proportion increased rapidly within 2 hours after burn and then increase moderately.4. Three-color analysis of cells was performed using a flow cytometer. The proportion of CD4+ and CD8+ T cells in MLNs was measured. the proportions of both CD4+ decreased in burn and sepsis groups. There was significant increases of CD8+ T cells at all time points. The ratio of CD4+/CD8+ was lowest on 72hours after burns. CD4+/CD8+ T cells ratio of dermabrasion group had a significant increase on postburn hours 72 (2.13±0.06versus 1.96±0.04, p<0.05).Conclusions:1. It's an ideal method to make lymphatic fistula model using oblique incision with microsurgical technique. Operative approach has little influence on the development of mesenteric lymphatic fistula obviously.2. CM-Dil marked bacteria is relatively sensitive, and it can reflect the microbial translocation condition all-round. Most of translocated bacteria can be killed by immune organ such as mesenteric lymph node. The level of bacteria and endotoxin in the lymph fluid is higher than blood of hepatic vein. However, the transport amount of the former is lower than the latter. In this way, the lymphatic pathway is an important rather than major pathway. 3. After burn, high-concentration microbe and endotoxin translocate to MLN and stimulate the lymph cells in MLN to secrete a lot of IL-18, IL-10, HMGB1; Meantime, much cytokine generated by bowel wall and local lymph nodes is recremented. Intestinal cytokine stimulate lymph nodes in gateway, activate lymph cells in the nodes and release new cytokine. Due to these three factors, the level of mediators of inflammation in intestinal lymph is strikingly higher than other tissue.4. T cell subsets in mesenteric lymph nodes after severe burn and balanced disorderly, the CD4+/CD8+ ratio decreased significantly, meanwhile the IL-18/IL-10 decreased too, which indicated the condition of immune suppression. Dermabrasion during shock stage can reduce the bacteria translocation and raise the ratios of CD4+/CD8+ T cells in the mesenteric lymph node.
|
PDF Full Text Request