| Object:Major thermal injury results in immunosuppression in patients and an increased susceptibility to subsequent septic complications and multiple organ failure. Numerous experimental studies have been directed at elucidating a better understanding of the mechanisms responsible for thermal injury-induced immunosuppression. The causative factors for immune dysfunction appear to be multiple aspects including T-lymphocyte dysfunction, increased levels of macrophage-derived inflammatory mediators and a cytokine imbalance toward a Th2 response. However, these studies only disclose the systemic immunity function change, the local immunity function change which caused by burn wound is unknown.The aim of this study was to determine the changes of cytokine levels including TNF-α, IFN-γand IL-4 in draining lymph fluid and the changes of CD4+, CD8+ and CD4+/CD8+ T cells ratio in draining lymph node of the burn wound.Methods:24 male adult Wistar rats were employed. Rats were anesthetized by intraperitoneal administration of pentobarbital (40mg/kg). The unilateral hind limb (burn limbs group, group A) of the rat was immersed in a water at 70℃for 30s randomly, which has been histologically shown to resulting in a deep second-degree burn of approximately 4% of the total body surface area. The contralateral hind limb (control limbs group, group B) underwent the same procedure except that the hind limb was immersed in a water bath of only 22℃for the same period. The "sham group" did not accept any treatment. On hours 6, 24 and 72 post burn, experiment was performed on anaesthetic rats under aseptic conditions in a standard veterinary operating theater. A midline abdominal incision was performed, after the intestines were displaced to the left, the right CILN was recognized in the right lower quadrant and a 1.5～2cm section of the efferent lymph trunk of CILN was isolated carefully under microscope, and the superior extremity of the isolated lymph vessel was ligated with 3/0 silk suture. Opposite side accepted the identical treatment. After the both hind limbs were moved about 30 minutes, the efferent lymph trunk of CILN swelled up, then punctured with a needle (diameter, 0.45mm), about 40μl lymph fluid was collected into eppendorf tube containing heparin (final concentration approximately 20IU/ml), then CILN were obtained. After lymph fluid was collected, lymph fluid was immediately centrifuged at 600 revolutions per minute for 10 min and the lymph plasma was stored in three aliquots at -80℃until analyzed. The lymph plasma was applied for the measurement of IFN-γ, IL-4 and TNF-αlevel by enzyme-linked immunosorbent assay, the CILN was for flow cytometric analysis. Result:Lymph fluid TNF-αconcentration in the efferent lymph trunk of CILN of the group A had a significant increase when compared to the group B on post burn hours 6, 24 and 72 (51.62±5.36 versus 17.84±1.58 p<0. 01, 27.35±2.57 versus 16.44±1.21 p<0. 01, 23.04±2.67 versus 17.24±2.05 p<0. 05, pg/ml). Lymph fluid TH2 cytokine IL-4 concentration of the group A had a significant increase when compared to the group B on post burn hours 6, 24 and 72 (14. 15±1.08 versus 8.97±0.72 p<0. 05, 19. 76 + 2. 15 versus 9.37±0.78 p<0.05, 14. 37 + 1. 73 versus 8. 80±1.24 p<0. 01, pg/ml), however, there were no statistically significant differences in the TH1 cytokine IFN-γ. There were statistically significant differences in IFN-γ/IL-4 ratio between the group A and group B on post burn hours 6 , 24 and 72 (2.27±0.34 versus 3.33±0.25 p<0.05, 1.54±0.19 versus 3.34±0.22 p<0. 05, 1.60±0.16 versus 2.52±0.24 p<0. 01). There were no statistically significant differences in the proportions of CD4+ T cells ratio on post burn hours 6 and 24 between the group A and group B (45.78±2.97% versus 48. 90±2. 39%, 43.42±1.50% versus 46.05±3.27%, p>0. 05), but a significant decrease on post burn wound infection hours 72 (38.55±2.26% versus 48.87±2.90%, p<0.05). There were no statistically significant differences in the proportions of CD8+ T cells ratio on post burn hours 6, 24 and 72 between the group A and group B (17.67±0.39% versus 18. 26±0.48%, 18. 31±1.12% versus 19. 00±0. 74%, 18. 28±0. 57% versus 17.95±0.74%, p>0.05). There were no statistically significant differences in CD4+/CD8+ T cells ratio between the group A and group B on post burn hours 6 and 24, but had a significant decrease on post burn hours 72 (2.13±0.16 versus 2. 68±0. 12, p<0. 05).Conclusion:1. Burn wound can increase TNF-αlevel of the draining lymph fluid.2. Burn wound can increase IL-4 level and decrease IFN-γ/IL-4 ratio of the draining lymph fluid and suppress the the local immunity function.3. Burn wound can decrease the CD4+/CD8+ T cells ratio of the draining lymph node and suppress the local immunity function. Object:Burn wound infection, one of the most serious complications of burn injury, can delay wound healing, lead to sepsis and increase mortality.Most researchers believe that bacteria and endotoxin may penetrate the capillaries of the affected tissues, invade blood and thus lead to bacteremia and endotoxemia. However, it is unclear whether bacteria and endotoxin of the urn wound can invade draining lymph node and draining lymph fluid. Moreover, burn wound infection results in immunosuppression. Numerous experimental studies have been directed at elucidating a better understanding of the mechanisms responsible for thermal injury infection-induced immunosuppression. The causative factors for immune dysfunction appear to be multiple aspects including T-lymphocyte dysfunction, increased or decreased levels of inflammatory factors. However, these studies only disclose the systemic immunity function change, the local immunity function change which caused by the pseudomonas aeruginosa of the burn wound is unknown.The aim of this study was to determine the lymphatic invasion route of bacteria and endotoxin of burn wound infected by pseudomonas aeruginosa and moreover, the effect of pseudomonas aeruginosa infection of the burn wound on the draining lymph node and lymph fluid.Methods:24 male adult Wistar rats were employed, Both hind limbs of the rat were immersed in a water bath at 95℃for 10 seconds respectively, which has been histologically shown to result in a full-thickness burn of approximately 8% of the total body surface area (4% each hind limb). After a waiting period of 15 minutes, 500μl bacterial inoculum (containing 1×10~8 CFU of total pseudomonas aeruginosa) was then applied subcutaneously into the burn site of the unilateral limb randomly, at the same time, 500μl 0.9% saline was applied subcutaneously into the burn site of the contralateral limb, they were divided into two groups: burn+infection limbs group (group A) and contrlateral limbs group (group B). The "sham group" (group C) did not accept any treatment. On hours 6, 24 and 72 post burn wound infection, experiment was performed on anaesthetic rats under aseptic conditions in a standard veterinary operating theater. A midline abdominal incision was performed, after the intestines were displaced to the left, the right CILN was recognized in the right lower quadrant and a 1. 5cm to 2cm section of the efferent lymph trunk of CILN was isolated carefully under microscope, and the superior extremity of the isolated lymph vessel was ligated with 3/0 silk suture. Opposite side accepted the identical treatment. After the both hind limbs were moved about 30 minutes, the efferent lymph trunk of CILN swelled up, then punctured with a needle (diameter, 0.45mm), about 40μl lymph fluid was collected into a pyrogen-free eppendorf tube containing heparin (final concentration approximately 20IU/ml), then CILN were obtained. After lymph fluid was collected, lymph fluid was immediately centrifuged at 600 revolutions per minute (rpm) for 10 min and the lymph plasma was stored in three aliquots at -80℃until analyzed. The lymph plasma was applied for the measurement of TNF-αlevels by enzyme-linked immunosorbent assay and endotoxin levels by Limulus Amebocyte Lysate test. The CILN was divided into two parts, one was for bacterial culture, another for flow cytometric analysis.Result:There was a higher invasion incidence in the group A when compared to the group B on post burn wound infection hours 6, 24 and 72 (100% versus 0% p<0.01, 100% versus 0% p<0.01, 100% versus 0% p<0.01). Endotoxin concentration of group A had a significant increase when compared to the group B on post burn wound infection hours 6, 24 and 72 (2.813±0. 116 versus 0. 316±0. 024, 2. 534±0. 252 versus 0. 287±0. 020 , 1. 382±0. 170 versus 0.276±0.022, P<0. 01). TNF-αconcentrations of the group A had a significant increase when compared to the group B on post burn wound infection hours 6, 24 and 72 (170.66±16. 18 versus 28.53±3.06, 131.43±10. 55 versus 27. 03±1. 93, 57. 63±6. 97 versus 19. 27±2. 29, pg/ml, P<0.01). There were no statistically significant difference in the proportions of both CD4+ and CD8+ T cells at all time points between the group A and group B. There were no significant differences in CD4+/CD8+ T cells ratio between the group A and group B on post burn wound infection hours 6 and 24, but a significant decrease on post burn wound infection hours 72 (1.99±0.12 versus 2.27±0.19, P<0.05).Conclusion:1. Bacteria and endotoxin of the burn wound can invade draining lymph node and lymph fluid.2. The pseudomonas aeruginosa infection of the burn wound can increase TNF-αlevel of the draining lymph fluid3. The pseudomonas aeruginosa infection of the burn wound can decrease CD4+/CD8+ T cells ratio of the draining lymph node. |
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