| Splanchnic ischemia and reperfusion is believed central to the pathogenesis of hemorrhagic shock-induced multiple organ failure (MOF), but the fundamental mechanism remains to be established. In the 1950s, Fine et al. proposed the absorption of endotoxin from the gut in conjunction with reduced hepatic detoxification as the basis for irreversible shock. In the 1980s extensive work by Deitch et al., Sori et al., and Alexander et al. revitalized interest in gut bacterial translocation and the portal vein became the focus for validating this unifying concept.In rodent model of mesenteric ischemia/reperfusion (I/R)-induced MOF, however, it was difficult to establish the role of gut bacterial translocation. Moreover, no documented bacteria or eridotoxin were found in the portal vein of severely injured patients who developed MOF. Recently, Magnotti et al. have redirected interest in the mesenteric lymph as the potential way conveying gut-derived proinflammatory agents responsible for distant organ injury. In a rodent model, they have shown that mesenteric lymph diversion prior to hemorrhagic shock abolishes lung injury. Furthermore, they showed that posthemorrhagic shock mesenteric lymph damages endothelial cells, whereas, the plasma from the portal veins from the same animals did not cause endothelial injury. In order to further establish mesenteric lymph as the mechanistic link to postshock MOF we sought to study its effects on neutrophils (PMNs). PMNs are key mediators in producing organ injury following severe injuryand shock. Consequently, We hypothesized that posthemorrhagic shock mesenteric lymph will prime PMNs for cytotoxicity as evidenced by enhanced superoxide production and increased surface expression of CD1 lb and CD18.7.5 % hypertonic saline resuscitation has been proved more effective than crystalloids in clinical work.The effect of 7.5%hypertonic saline (HTS) on resuscitation is prompt, effective to recover and maintain the blood pressure level in certain period. Some basic researches showed that HTS resuscitation can prevent the damage of barrier effection of the intestine, bacterial translocation, adhesion molecule expression on endothelial cell, and limit lung injury or MOF. Therefore, the relations between HTS and the effections of posthemorrhagic mesenteric lymph on neutrophil activationas become the goal of our study.In this study we want to explore and discuss the role of mesenteric lymph after trauma-hemorrhagic shock on neutrophil activation, as well as the influence of HTS resuscitation on neutrophil activation, so that we maybe find out the potential relation between mesenteric lymph after trauma-hemorrhagic shock and shock induced MOF and provide basic evidence and effective methods to prevent or treat shock induced MOF in clinical work.Part I PMN adhesion molecule expression after trauma-hemorrhagic shockForty eight healthy male Sprague-Dawley rats were randomized into eight groups: control group, T/SS (Trauma/ sham shock) group, groups of T/HS with different fluid resuscitation such as Lactated Ringers, HTS and blood, and groups of T/SS with resuscitation by different fluids. Rats were anesthetized by sodium pentobarbital (50 mg/kg, i.p.), right subcostal laparotomy (trauma) was followed. A femoral artery catheter (22 gauge, BD) was placed for monitoring blood pressure and drawing blood, and an internal jugular vein catheter (24 gauge, BD) was placed for fluid injection. The rats were then subjected to hemorrhagic shock by drawing blood until their mean arterial pressure (MAP) reached 40 mmHg. The blood pressure was maintained at 40 mmHg for 90 min by withdrawing or reinfusing, the shed blood was kept at 37°C as required. At the end of the 90-min shock or sham shock without drawing blood period,the animals were resuscitated with either Ringer's lactate (3 volumes of shed blood), 7.5% hypertonic saline (0.364 volume of shed blood) or same volume of shed blood. The T/SS group received an equivalent volume of different fluid. The total sodium load for each group was held constant. Whole blood samples for the neutrophil CDl lb and CD 18 assays were collected before shock as well as at the end of shock period, and at end of 1st and 2nd hour after the resuscitation.Results: The MAP between different group after resuscitation has no significant difference (P>0.05).There was no significant difference of PMN adhesion molecule expression comparing with the groups of control, T/SS and T/SS resuscitated with different fluid. Comparing with T/SS groups, the PMN adhesion molecule expression has significant increased at the end of shock period in all shock groups(P<0.05). Comparing with that at end of shock period, the PMN adhesion molecule expression during resuscitation in the shock group resuscitated with Ringer's lactate was significant elevated (P<0.05), however, the PMN CD 18 expression of during resuscitation with HTS has significant decreased(P<0.05), the PMN CDl 8 and CDl lb expression of during resuscitation with blood has significant decreased(P<0.05)Part II Gut-Derived mesenteric lymph activates PMN(1) The influence of ligation of mesenteric lymph duct on neutrophil activation. The groups of T/HS without duct ligation, Pre-T/HS duct ligation, Post-T/HS duct ligation, and their T/SS were included. In shock groups: either mesenteric lymph duct was ligated first in group of Pre-T/HS duct ligation or no duct ligation in group of T/HS without duct ligation , then was subjected to T/HS, before resuscitation by Ringer's lactate, lymph duct was ligated in the group of Post-T/HS duct ligation, then they and their T/SS were resuscitated with Ringer's lactate (3 volume of shed blood). Femoral artery blood samples were collected as above, the PMN adhesion molecule expression was examined.(2) The ability of Post-T/HS mesenteric lymph to prime PMN The groups included T/SS, T/HS and its T/SS resuscitated with Ringer's lactate. The mainmesenteric lymphatic was carmulated with 24G catheter. Lymph samples were collected during preshock in one hour, the first resuscitation hour into heparin-wetted iced tuberculin syringes. Post-T/HS mesenteric lymph-induced rat PMN adhesion molecule expression and respiratory burst activity was examined using a FACScan flow cytometer.(3) The ability of Post-T/HS plasma of portal vein to prime PMN Three groups were included as above. Portal vein plasm samples were collected before shock, at end of the first resuscitation in shock and sham shock groups. Post-T/HS portal vein plasma-induced rat PMN adhesion molecule expression and respiratory burst activity were examined as above. Results: There was no significant difference of expressions of CD 18 and CDllb in all sham shock with or without ligation of mesenteric lymph duct (P>0.05). It showed that ligation of mesenteric lymph duct itself had no effect on the expression of PMN CD 18 or CDllb. Comparing with the group of T/HS without duct ligation, the expressions of PMN CD 18 and CDllb at end of shock and during resuscitation in the group of pre-T/HS duct ligation were significantly decreased (P<0.05). It was suggested that ligation of mesenteric lymph duct before shock prevent PMN activation at shock and resuscitation period. Comparing with the group of T/HS without duct ligation, the expressions of PMN CD 18 and CDllb during resuscitation in the group of post-T/HS duct ligation were significantly decreased (P<0.05). It was suggested that ligation of mesenteric lymph duct after shock and before resuscitation prevent PMN activation at resuscitation period.The expression of rat PMN CD18 and CDllb after incubating with post-T/HS mesenteric lymph was significantly increased (PO.05), comparing with all T/SS groups. However, rat PMN CD18 and CDllb expression after incubating with portal vein plasma was no significate difference (P>0.05). Neutrophil respiratory burst activity test of post-T/HS lymph and portal plasma showed same results as above. This means portal vein plasma has the ability to activate neutrophils but not portal plasma.Partm Hypertonic saline resuscitation limites mesenteric lymph-induced PMN activation after T/HSSeven groups were included, with 6 rats in each group. They were groups of rats subjected to T/SS, rats subjected to T/SS or T/HS resuscitated with Ringer's lactate, HTS or blood. Measuring the volume of mesenteric lymph collected before shock, at end of the first and second resuscitation in shock and sham shock groups. In this experiment we used the mesenteric lymph during the two hour resuscitation to check the ability of post-T/HS mesenteric lmph to prime PMN. Post-T/HS mesenteric lymph-induced human and rat PMN adhesion molecule expression and respiratory burst activity was examined by FACScan flow cytometer as above. Results: Comparing with their T/SS groups, the volume of mesenteric lymph during resuscitation collected from T/HS group resuscitated with different fluids was significantly increased (PO.05);comparing with that of T/HS group resuscitated with Ringer's lactate (RL), the volume of mesenteric lymph during resuscitation collected from T/HS group resuscitated with HTS or blood was significantly decreased (PO.05). Comparing with that of their T/SS groups, the rat and human PMN CDllb and CD 18 expression induced by Post-T/HS mesenteric lymph of resuscitation with Ringer's lactate was significantly increased (P<0.05), however, the rat and human PMN CDllb and CD18 expression induced by Post-T/HS mesenteric lymph of resuscitation with HTS or blood was no significantly increased (P>0.05). Comparing with that of Post-T/HS mesenteric lymph of resuscitation with Ringer's lactate, the ability of Post-T/HS mesenteric lymph of resuscitation with HTS or blood to induce rat and human PMN CDllb and CD 18 expression was significantly decreased (P<0.05). Neutrophil respiratory burst activity test of post-T/HS lymph of resuscitation with different fluid showed same results as above.ConclusionTrauma hemorrhagic shock induces neutrophil activation. Shock-induced neutrophil activation is prevented by mesenteric lymph duct ligation. Posthemorrhagic shock mesenteric lymph but not portal plasma primes circulating neutrophils.7.5% hypertonic saline resuscitation limits neutrophil activation after trauma-hemorrhagic shock, decreases the flow of mesenteric lymph, and the ability of mesenteric lymph to induce PMN adhesion molecule expression and superoxide production. |
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