Role Of Post-hemorrhagic Shock Mesenteric Lymph Enhancing Vascular Permeability

Vascular hyperpermeability is an important reason of infractoryhypotension, multiple organ dysfunction syndrome (MODS), and multipleorgan failure (MOF) induced by severe shock. Therefore, the effectiveprevention and treatment of vascular hyperpermeability are the key link tothe therapy of severe hemorrhagic shock. There was an important role ofintestinal lymphatic pathway during the multiple organ injury followinghemorrhagic shock. Blockade of post-hemorrhagic shock mesentericlymph (PHSML) return to systemic circulation, using the methods ofmesenteric lymph duct ligation and mesenteric lymph drainage, coulddecrease the imflammatory response and structural damage of liver,kidney, heart, lung, etc. However, the relationship between the PHSMLreturn and vascular hyperpermeability following hemorrhagic shock,needs further study. Therefore, in this study, we investigated the role ofPHSML on vascular hyperpermeability following hemorrhagic shock.First, eighteen male Wistar rats were randomized to the sham, shock,shock+drainage groups. All of the animals were anesthetized byintramuscular injection with pentobarbital. The right femoral vein wasseparated and cannulated for anticoagulation with heparin sodium. Aminimally heparinized polyethylene catheter was introduced into the rightfemoral artery for continuously monitoring the animals’ mean arterypressure (MAP) through the experiment. Another catheter was insertedinto the left femoral artery for bleeding by a syringe pump. A laparotomywas carried out to separate the mesenteric lymph duct from surrounding connective tissues. After a30-min stabilization period, the animals weresubjected to sham, hemorrhagic shock and hemorrhagic shock plusmesenteric lymph drainage. In the shock, shock+drainage groups, bloodwere withdrawn from the right femoral artery at an even speed. The MAPwas reduced to40mmHg within10min and maintained at this level bywithdrawing or reperfusing shed blood as needed, thus, the hemorrhagicshock model was established. After90min of hypotension, the ejectiveblood and the equal Ringer’s solution were reperfused within30minthrough right femoral vein. The observation continue6h after the end ofinfusion. In the shock+drainage group, the mesenteric lymph duct wascannulated and mesenteric lymph was drained up to6hours after infusionfinished. In the sham group, the rats were anesthetized, cannulated andoperated as described above, but no blood was withdrawn or infused.At5.5h after infusion finished or corresponding time, the EBsolution of1%was injected with a dose of30mg/kg through femoral vein.30min later, the lavage of systemic circulation and pulmonary circulationwas performed using normal saline. Then, the fixed position tissues ofmyocardium, liver, spleen, kidney, intestine, and lung were take out forthe observation of color in appearance, the detections of EB concentrationin tissue and the ratio of dry weight and wet weight (D/W). The currentresults showed that the EB concentrations of myocardium, liver, spleen,kidney, intestine, and lung in the shock group, and myocardium, andkidney in the shock+drainage group all were significantly increased thanthat of the sham group. Moreover, the EB concentrations of myocardium,liver, spleen, kidney, intestine, and lung in the shock+drainage group weresignificantly decreased compared with the shock group. In addition, theratios of D/W of myocardium, liver, spleen, kidney, intestine, and lung inthe shock group were lower than those in the sham group, and theseindices in the shock+drainage group were higher than those in shockgroup. These findings indicated that there was a vital role of PHSML drainage reducing the vascular permeability in tissues followinghemorrhagic shock.Afterwards, human umbilical vein endothelial cells (HUVECs) wereused to next experimental material, and were incubated with the PHSMLin vitro, to observe the effect of PHSML on cellular permeability ofHUVECs. According to the different treatment factors, experiment in thispart was divided into seven groups. As follows: DMEM group(Dulbecco’s modified eagle’s medium, DMEM), DMEM+FBS (DMEMand10%(v/v) fetal bovine serum), LPS group (DMEM and10μg/mlLPS),4%PHMSL0~3h group (DMEM and4%(V/V) PHMSL obtainedfrom shocked rats at0~3h after resuscitation),10%PHMSL0~3h group(DMEM and10%(V/V) PHMSL obtained from shocked rats at0~3hafter resuscitation),4%PHMSL3~6h group (DMEM and4%(V/V)PHMSL obtained from shocked rats at3~6h after resuscitation), and10%PHMSL3~6h group (DMEM and10%(V/V) PHMSL obtained fromshocked rats at3~6h after resuscitation). After6h of co-culture withdifferent treatment fators, the cellular morphology and viability ofHUVECs were observed by the inverted microscope and the mothods ofMTT, respectively. Meanwhile, the trans-endothelial electrical resistance(TEER) of HUVECs and permeability of FITC-Albumin were observedusing the costar transwell system. The results showed that there were noobvious differences in morphology, viability, TEER, and permeability ofFITC-Albumin between the DMEM and FBS+DMEM groups. The4%PHMSL of0~3h,10%PHMSL of0~3h,4%PHMSL of3~6h,10%PHMSL of3~6h, and LPS all caused the damage of HUVECs, decreasethe viability and TEER of HUVECs, and increased the permeability ofHUVECs. Furthermore, to investigate the mechanism of PHMSLenhancing the permeability of HUVECs, we observed the effects ofdifferent treatment factors above on the expressions of F-actin, acytoskeletal protein, of HUVECs, with the method of fluorescent cytochemistry staining. The results showed that the4%PHMSL of0~3h,10%PHMSL of0~3h,4%PHMSL of3~6h,10%PHMSL of3~6h, andLPS all decreased the expression of F-actin in HUVECs.In summary, these current results in this study indicated thatmesenteric lymph drainage was beneficial to reduce the hyper-vacularpermeability in multiple organs induced by hemorrhagic shock;meanwhile, the PHSML in vitro could increase the cellular permeability ofHUVECs, by which decreased the expressions of F-actin. The presentfinding provides an experimental evidence for targeting the return ofmesenteric lymph and aiming at the vascular permeability to prevent andtreat critical shock. However, the screening of toxic substances in post-hemorrhagic shock mesenteric lymph and mechanism of vascularhyperpermeability caused by PHSML need further investigation.