Role of Rho kinase in blocking the return of mesenteric lymph to improve vascular calcium sensitivity in hemorrhagic shock rats

AIM: To observe the role of Rho kinase in mesenteric lymph duct ligation or mesenteric lymph drainage to improve vascular calcium sensitivity in the rats subjected to hemorrhagic shock. METHODS: Male Wistar rats were randomly divided into sham group, shock group, shock+ligation (shock plus mesenteric lymph duct ligation) group and shock+drainage (shock plus mesenteric lymph drainage) group. After induction of shock (hypotension at 40 mmHg) for 3 h, the vascular rings of superior mesenteric artery (SMA) were prepared and used to measure the response to gradient calcium ions for determining the calcium sensitivity with a wire myograph system. In shock+ligation group and shock+drainage group, the vascular rings were incubated with Rho kinase agonist angiotensinⅡ or antagonist fasudil before the measurement of the response to gradient calcium ions. RESULTS: The calcium sensitivity of vascular rings in shock group was significantly lower than that in sham group, and that in shock+ligation group and shock+drainage group was significantly higher than that in shock group, but still lower than that in sham group. AngⅡ elevated the contractile activity of the vascular rings in response to gradient calcium ions and the pD₂, and fasudil significantly decreased the response to gradient calcium ions and E_max in shock+ligation group and shock+drainage group. At the same time, fasudil decreased the pD₂ in shock+ligation group. CONCLUSION: Rho kinase plays an important role in blocking shock mesenteric lymph return that improves calcium sensitivity.     

Mesenteric lymph drainage alleviates spleen injury in hemorrhagic shock rats

AIM：To observe the effects of post-shock mesenteric lymph (PSML) drainage on histopathology, apoptosis, cell cycle and proliferation of the spleen in rats with hemorrhagic shock. METHODS：Eighteen Wistar rats were randomly divided into sham, shock and shock+drainage groups (n=6 in each group). The hemorrhagic shock model was established in the shock and shock+drainage groups. Fluid resuscitation for 30 min was performed 1.5 h after hypotension, and PSML was drained in the rats in shock+drainage group from 1 h after hypotension to 3 h after resuscitation finished. The fixed spleen tissue was harvested from each rat for histological observation with HE staining. The apoptosis of splenocytes was observed by Hoechst 33258 staining. The expression of Bcl-2 and Bax proteins was detected by immunohistochemical staining. The cell cycle and the expression of p53 protein were measured by flow cytometry, and the proliferation index (PI) was calculated. RESULTS：Compared with sham group, splenic tissue injury appeared in the shocked rats. The apoptotic cells and the expression of Bax and p53 in shock group were increased, while Bcl-2 expression was decreased. The percentage of G2/M cells in shock group was decreased. Compared with shock group, the splenic tissue damage in shock+drainage group was significantly attenuated. Moreover, the number of apoptotic cells, the percentage of G0/G1 cells, and the expression of Bax and p53 were obviously decreased, and the G2/M cells, Bcl-2 protein expression and PI were significantly increased in shock+drainage group. CONCLUSION: PSML drainage alleviates splenic injury in hemorrhagic shock rats, which may be related to reducing the apoptosis of splenocytes.     

Role of hydrogen sulfide in alleviation of liver injury by mesenteric lymph drainage in hemorrhagic shock rats

AIM：To investigate the role of hydrogen sulfide (H2S) in alleviation of liver injury by mesenteric lymph drainage in hemorrhagic shock rats. METHODS：A hemorrhagic shock model was established in male Wistar rats. DL-propargylglycine (PPG), an inhibitor of cystathionine γ-lyase (CSE) which is a synthase of H2S, or sodium hydrosulfide (NaHS), a donor of H2S, was administered to the hemorrhagic shock rats with mesenteric lymph drainage. The rats were randomly divided into sham, shock, shock+drainage, shock+drainage+PPG (45 mg/kg, ip, 0.5 h before hemorrhage) and shock+drainage+NaHS (28 μmol/kg, ip, 0.5 h before hemorrhage) groups. Fluid resuscitation was performed 1 h after hypotension, and then mesenteric lymph was drained in the rats of shock+drainage, shock+drainage+PPG and shock+drainage+NaHS groups for 3 h. The hepatic histomorphology was observed. The biochemical indexes of hepatic function in plasma, and H2S, CSE, Toll-like receptor 4 (TLR4), interleukin (IL)-10, IL-12 and tumor necrosis factor α (TNF-α) in hepatic homogenate were also examined. RESULTS：The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bile acid (TBA) in plasma, and H2S, CSE, TLR4, IL-10, IL-12 and TNF-α in hepatic homogenate in shock group were significantly higher than those in sham group. Mesenteric lymph drainage obviously reduced these indexes in shock rats, except for TLR4. PPG further decreased these indexes except for CSE, while NaHS increased these indexes except for TBA and CSE. Morphological observation showed that liver injury appeared in the rats from shock and shock+drainage+NaHS groups, and there was nearly normal hepatic structure in the rats from sham, shock+drainage and shock+drainage+PPG groups. CONCLUSION：The mechanism of mesenteric lymph drainage alleviating liver injury in hemorrhagic shock rats is related to reducing the production of H2S and alleviating the H2S-mediated inflammation.     

Intravenous injection of mesenteric lymph from shock rats damages red blood cells in normal rats

AIM: To observe the effects of intravenous injection of the mesenteric lymph from shock rats on the characteristics and metabolism of red blood cells (RBC), and blood viscosity in normal rats. METHODS: The mesenteric lymph samples, collected from the rats 1 to 3 h after hemorrhagic shock, centrifuged to remove all cellular components and diluted with equal volume of saline, were intravenously injected into normal rats at dose of 2 mL/kg through femoral vein within 30 min. The equal volume of saline was intravenously injected into other normal rats as controls. At 2.5 h after injection, the blood samples were collected from the abdominal aorta for determining the routine parameters, adenosine triphosphate (ATP), lactic acid (LA), 2, 3-diphosphoglycerate (2, 3-DPG), ion concentrations of intra-and extracellular fluid of the RBC and blood viscosity. RESULTS: Intravenous injection of shocked mesenteric lymph reduced the number of RBC, the concentration of hemoglobin, the hematocrit and the content of ATP. Intravenous injection of shocked mesenteric lymph significantly increased the mean corpuscular volume (MCV), 2, 3-DPG, LA in RBC and the whole blood reduced viscosity. However, no obvious effect of the injection on ion concentrations of intra-and extracellular fluid of RBC, whole blood viscosity and plasma viscosity was observed. CONCLUSION: Intravenous injection of shocked mesenteric lymph causes the disorders of energy metabolism in RBC, thus increasing the MCV and whole blood reduced viscosity. Shocked mesenteric lymph damages RBC.     

Role of post-hemorrhagic shock mesenteric lymph in enhancement of vascular permeability

AIM：To investigate the role of post-hemorrhagic shock mesenteric lymph (PHSML) in the enhancement of vascular permeability. METHODS：Eighteen Wistar rats were randomized into sham group, shock group, and shock plus mesenteric lymph drainage (shock + drainage) group. The rats in shock group and shock + drainage group were routinely subjected to hemorrhagic shock and hypotension ［(40±2） mmHg］ was maintained for 90 min, and then the fluid resuscitation was performed. Mesenteric lymph was drained in the rats in shock+drainage group from resuscitation finished to 6 h, for the observation of PHSML drainage on the vascular permeability in multiple tissues of hemorrhagic shock rats. Afterwards, human umbilical vein endothelial cells (HUVECs) were incubated with the PHSML in vitro to observe the effects of PHSML on the morphology and permeability of HUVECs. RESULTS：The degree of blue color and concentrations of Evens blue in the lung, myocardium, kidney, liver, spleen and small intestine were significantly increased in the shocked rats than that in sham group, while the ratios of the dry weight to the wet weight were decreased. The mesenteric lymph drainage reversed these changes. Meanwhile, 4% and 10% of PHSML at 0~3 h and 3~6 h after resuscitation, and lipopolysaccharide (10 mg/L) all caused the damage of HUVECs, decreased the viability and trans-endothelial electrical resistance of HUVECs, and increased the permeability of HUVECs to fluorescein isothiocyanate-labeled albumin. CONCLUSION：PHSML is a vital factor in the enhancement of vascular permeability.     

Effect of normal mesenteric lymph on multiple organ injury in mice with endotoxic shock

AIM: To observe the effects of normal mesenteric lymph (NML) on the lung, heart and liver injuries and the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) in the mice with endotoxic shock (ES). METHODS: The NML was drained form health male BALB/c mice for the intervention of ES after the removal of cellular constituent. Lipopolysaccharide (LPS, 35 mg/kg) was intraperitoneally injected into the mice for the establishment of ES model. After 60 min of LPS injection, the administration of NML (1/15 of whole blood volume) was performed through the femoral artery in NML+ES group. Meanwhile, the mean arterial pressure (MAP) was monitored during the experiment. At 6 h after intraperitoneal injection of LPS or the corresponding time point, blood samples were harvested from the heart through apical centesis for determination of the biochemical indexes to reflect myocardial and hepatocyte injuries. Simultaneously, the lung, heart and liver tissue specimens from a fixed location were harvested for the observation of histomorphology and the measurement of phosphorylation levels of p38 MAPK, ERK1/2 and JNK. RESULTS: Compared with sham shock (SS) group, MAP in ES group and NML+ES group remarkably decreased at multiple time points after intraperitoneal injection of LPS. However, MAP in NML+ES group at 80 min, 90 min, 190 min, 210 min, 240 min, 250 min, 340 min, 350 min, and 360 min were significantly increased compared with ES group. There were normal structures in the lung, liver and myocardium of the mice in SS group, while the morphological damages of these tissues appeared in ES group. Meanwhile, the damages were attenuated in the mice of NML+ES group. The activities of AST, ALT and CK-MB in the plasma in ES group were remarkably higher than those in SS group. The CK-MB activity in NML+ES group was also increased compared with SS group, and the activities of AST and LDH-1 were lower than those in ES group. At 6 h after LPS injection, the phosphorylation levels of p38 MAPK, ERK1/2 and JNK in the lung tissues were remarkably increased. Meanwhile, no statistical difference of these indexes between the myocardial and hepatic tissues was observed. NML intervention decreased the phosphorylation levels of p38 MAPK in the lung tissues, and p38 MAPK, ERK1/2 and JNK in the myocardial tissues. CONCLUSION: The NML administration alleviates multi-organ injuries and reduces the phosphorylation level of p38 MAPK in the lung tissues in the mice subjected to ES.     

Effects of hemorrhagic shock on BKCa channel tyrosine phosphorylation level in mesenteric artery in rats

AIM: To study the effects of hemorrhagic shock on BK_Ca channel tyrosine phosphorylation in rat superior mesenteric artery and the role of nitric oxide (NO) in BK_Ca channel tyrosine phosphorylation. METHODS: The hemorrhagic shock model [(35±5)mmHg] was established in rats and the whole lysate of superior mesenteric artery were extracted and analyzed by immune precipition (IP) and immunoblotting. The tyrosine phosphorylation levels of BK_Ca channel alpha-subunit in mesenteric artery in hemorrhagic shock rats were investigated, and the modulation of BK_Ca channel alpha-subunit tyrosine phosphorylation by NO and its dose-and time-dependended relationships were observed. RESULTS: The tyrosine phosphorylation level of BK_Ca channel alpha-subunit in mesenteric artery in rats increased significantly after hemorrhagic shock 2 h and 4 h (P<0.01 ), and L-arginine (5×10^-5-5×10^-4 mol/L) up-regulated BK_Ca channel alpha-subunit tyrosine phosphorylation in primary cultured VSMC in a 30 min incubation and without significant decrease after 2 h; L-arginine induced BK_Ca channel alpha-subunit tyrosine phosphorylation in a dose-dependent manner. CONCLUSION: Hemorrhagic shock enhances BK_Ca channel tyrosine phosphorylation in resistant artery in rats, and NO is involved in this process.     

Mechanism of calcium sensitivity in lymphatic hypo-reactivity in hemorrhagic shock rats

AIM: To observe the changes of lymphatic reactivity to norepinephrine (NE) and calcium sensitivity in vitro in hemorrhagic shock (HS) rats. METHODS: Male Wistar rats were randomly divided into sham group (with only operation), HS group (duplicating HS model, and divided into shock 1 h and shock 2 h subgroups). The thoracic duct rings (n=48 in each group) were prepared for assaying the lymphatic reactivity to NE and calcium sensitivity by lymphatic tension measurement technique in vitro with isolated perfusion system. Meanwhile, the effects of angiotensin Ⅱ (Ang Ⅱ) and insulin (Ins) on lymphatic reactivity were also observed. RESULTS: Compared with sham group, the NE concentration-response curves in HS 1 h and HS 2 h groups, and calcium concentration-response curves in HS 2 h group were obviously shifted to right. The lymphatic reactivity to NE, contraction to calcium, maximum effect(E_max)and avidity index (pD₂) were markedly reduced. In HS group, after incubating with calcium sensitizer Ang Ⅱ, the lymphatic reactivity to NE and calcium sensitivity were significantly increased but reduced in sham group. However, calcium sensitivity inhibitor Ins decreased the lymphatic contractile response to NE and Ca²⁺. CONCLUSION: The lymphatic hypo-reactivity in hemorrhagic shock rats is related to calcium desensitization, indicating a mechanism of lymphatic hypo-contraction.     

Effect of intestinal lymphatic pathway on apoptosis of lung tissue in rats by two-hit

AIM: To observe the effects of mesenteric lymph duct ligation on apoptosis of lung tissue,correlated gene expression with apoptosis and TNF-α,IL-6 contents in rats by two-hit.METHODS: 45 Wistar rats were divided into three groups: the ligation group,the non-ligation group and sham group,and the two-hit model was established by means of hemorrhage and LPS treatments.Ligating mesenteric lymph duct was conducted in ligation group.After 24 hours,the pathological sections of lung tissue were prepared for determining the apoptosis rate by TUNEL method and expressions of Bcl-2 and Bax were observed by immunohistochemical test.The lung homogenate was also prepared for determining the contents of TNF-α and IL-6 by ELISA.RESULTS: After two-hit,the apoptosis rate,Bax expression in lung tissue and contents of TNF-α and IL-6 in serum and lung homogenate in non-ligation group were increased and Bcl-2 expression was lower than that in sham group and ligation group significantly (P<0.01,P<0.05).Apoptosis rate in ligation group was no statistics difference with sham group (P>0.05),and the expression of Bcl-2 protein was increased and Bax was lower than that of sham group (P<0.01,P<0.05).CONCLUSION: Blockage of intestinal lymphatic pathway reduces the apoptosis of lung in two-hit rats,and its mechanism might relate to reduced the levels of TNF-α and IL-6 and improved the expression of Bcl-2 protein in lung by the ligation of mesenteric lymph duct.The mesenteric lymph of two-hit might play an important role in the pathogenesis of acute lung injury (ALI).     

Isolation of exosomes and effect of stellate ganglion block on the number of exosomes in post-hemorrhagic shock mesenteric lymph of rats

AIM To isolate the exosomes in mesenteric lymph, verify the source of exosomes, and observe the effect of stellate ganglion block （SGB） on the number of exosomes in post-hemorrhagic shock mesenteric lymph （PHSML） of rats. METHODS Twenty-four male rats were randomly divided into sham, sham+SGB, shock, and shock+SGB groups. SGB was performed before the establishment of hemorrhagic shock model using the routine methods in our lab. The PHSML was drained for exosomes isolation. The exosomes were identified through particle size analysis and CD63 protein expression. The expression of epithelial cell adhesion molecule （EpCAM） was detected to identify whether the exosomes were derived from epithelial cell. The number of exosomes in various mesenteric lymphs was measured using the flow cytometry. RESULTS The diameter of granular material extracted from mesenteric lymph was about 100 nm. The positive expression of exosomes pecific protein CD63 indicated the successful isolation of exosomes, and the EpCAM expression verified the exosomes were derived from intestinal epithelial cells. The number of exosomes in mesenteric lymph isolated from the rats of Shock group was obviously increased compared to that from the Sham group （P<0.05）, while the exosomes from the Shock+SGB group was markedly decreased when compared to Shock group （P<0.05）. CONCLUSION The current study establishes the isolation technique of exosomes in mesenteric lymph, and proved the exosomes were derived from the intestinal epithelial cells. SGB treatment reduces the number of exosomes in PHSML.     

Mechanism of vascular hyporeactivity induced by NO in hemorrhagic shock

AIM: To investigate the role of nitric oxide (NO) in vascular hyporeactivity during prolonged hemorrhagic shock (HS). METHODS: Anesthetized Sprague-Dawley rats (180-220 g) were subjected to HS insult in which they were bled to a mean arterial pressure (MAP) of 40 mmHg (5.33 kPa) and arteriolar reactivity to norepinephrine in spinotrapezius was detected. The constant MAP of 40 mmHg was maintained until vascular hyporeactivity had occurred and then were resuscitated or sacrificed for further analysis. NO synthase (NOS) activity was measured ex vivo by the conversion of [³H]-arginine to [³H]-citrulline in homogenates from heart, lung, liver, spleen, duodunum, skeletal muscle. 24 h survival rates of resuscitated rats were observed with and without administration of aminoguanidine (AG), a selective inducible NOS (iNOS) inhibitor. Mesenteric arteriolar smooth muscle cells (ASMC) were isolated, and the effects of L-arginine (L-Arg) on membrane potential (MP) of ASMC were determined by fluorescent probe and confocal microscopy in the absence and presence of AG. RESULTS: When vascular hyporeactivity occurred, an increase of NOS activity was observed in liver and heart. Resuscitated rats with AG had a higher survival rate compared with that of control. The MP of ASMC was decreased (more negative) immediately following the addition of L-Arg, and the hyperpolarization effects of L-Arg were partially blocked in the presence of AG. CONCLUSION: These results suggest that excessive NO produced in HS is responsible for the occurrence of vascular hyporeactivity in prolonged hemorrhagic shock, and one of the mechanisms of which may be hyperpolarization of ASMC caused by NO.     

Estrogen treatment enhances mesenteric lymphatic contractility in rats after hemorrhagic shock

AIM To investigate the effects of 17β-estradiol （E2） treatment on the mesenteric lymphatic microcirculation and isolated lymphatic contractility in rats after hemorrhagic shock, and to explore the relationship between contractility and the difference between intra- and extracellular calcium ion concentrations （［Ca²⁺］） of lymphatic smooth muscle cells （LSMCs）. METHODS Male Wistar rats were divided into sham group, shock group and shock+E2 group. The rats were subjected to hemorrhage ［（40±2） mmHg for 90 min］ and resuscitation with or without subcutaneous injection of E2 （2 mg/kg）. After resuscitation for 3 h, the mesenteric lymphatic microcirculation in vivo was observed. Moreover, the isolated mesenteric microlymphatic rings were prepared for the observations of lymphatic contractility evaluated by the indexes including end-systolic diameter, end-diastolic diameter, contraction frequency （CF） and passive diameter. Meanwhile, the difference between intra- and extracellular ［Ca²⁺］ of LSMCs was recorded during lymphatic contraction. RESULTS Treatment with E2 significantly enhanced the CF, total contractile fraction and lymphatic dynamics index in vivo in the rats after hemorrhagic shock, and increased the CF, the fractional pump flow and the difference between intra- and extracellular ［Ca²⁺］ of LSMCs in isolated lymphatics from the shocked rats （P<0.05）. CONCLUSION Estrogen treatment enhances lymphatic contractility in rats after hemorrhagic shock, which is related to enhancement of difference between intra- and extracellular ［Ca²⁺］ of LSMCs.     

Hypertonic NaCl-NaAc improves the microcirculation in rats subjected to hemorrhagic shock

AIM: To study the effects of hypertonic NaCl-NaAc on microcirculation in hemorrhage-shocked rats.METHODS: SD rats were randomized into three groups of 7.5% NaCl(hypertonic saline, HS), 5% NaCl-3.5% NaAc(hypertonic sodium acetate, HSA) and 0.9% NaCl(normal saline, NS). 4 mL/kg HS, HSA or NS was given intravenously following hemorrhagic shock. The microcirculation of spinotrapezius muscle was observed.RESULTS: HS increased mean aortic pressure more significant than HSA. Variables including arteriolar and venular diameter, velocity and volumetric flow rate and open capillaries were increased and erythrocyte aggregation was decreased in 5 min after resuscitation with both HS and HSA solutions.5 min later, variables were deteriorated in HS group.After local treatment, arteriolar and venular diameters were dilated significantly in HSA group.CONCLUSION:HSA had superior effects to HS in improving microcirculation of hemorrhage-shocked rats.     

Effect of mesenteric lymph duct ligation on actue lung injury in rats

AIM：To explore the effect of mesenteric lymph duct ligation against actue lung injury (ALI) in rats.METHODS：45 Wistar rats were divided into three groups：the ligation group,the non-ligation group and sham operated group,and the two-hit model was established by hemorrhage and LPS injection.Mesenteric lymph was diverted by ligating mesenteric lymph duct in ligation group.All rats facilitated blood withdrawal for blood sample to arterial gas analysis after 24 hours.Then the WBC,NO,NOS,MDA,SOD and lung permeability index (LPI) were determined in bronchoalveolar lavage fluid (BALF),the MPO and ATPase activity were determined in lung homogenate.The ultrastructure was also observed.RESULTS：After two-hit,the PaCO2,the total cells and PMN,the NO2-/NO3-,NOS and MDA content in BALF and MPO activity in lung homogenate and LPI in non-ligation group were significantly increased than those in sham operated group.PaO2 and pH in arterial blood,SOD in BALF and the ATPase in lung homogenate were significantly lower (P<0.01 or P<0.05).The total cells and PMN,MDA,NO2-/NO3- in BALF,LPI in ligation group were significantly increased than those in sham operated group,and SOD in BALF was significantly lower (P<0.01 or P<0.05).The pH and PaO2 in arterial blood,the ATPase in lung homogenate in ligation group were significantly increased than those in non-ligation group,and the PaCO2,the total cells,PMN,NO2-/NO3-,NOS,MDA in BALF,LPI,and MPO in lung homogenate in ligation group were significantly lower than those in non-ligation group (P<0.01 or P<0.05).The injury of pulmonary vascular endothelium in ligation group was lighter than that in non-ligation group.CONCLUSION：The ligation of mesenteric lymph duct attenuates the ALI of rats.Mesenteric lymph might play an important role in the pathogenesis of ALI.     

Substance P enhances pump function of isolated lymphatics from hemorrhagic shock rats

AIM: To establish a technique of lymphatic perfusion in vitro and to determine the effect of substance P (SP) on lymphatic contractility during the process of hemorrhagic shock (HS). METHODS: Male Wistar rats were randomly divided into control group (surgical procedure only) and HS group . Thoracic ducts were isolated from HS rats at the corresponding time points. The segment of thoracic duct was prepared, perfused in vitro at the transmural pressure of 3 cmH₂O and stimulated with gradient concentrations of SP to measure the lymphatic contractile activity. The end-systolic diameter, end-diastolic diameter, contraction frequency (CF) and passive diameter of isolated lymphatics were measured. The contraction amplitude (CA), tonic index (TI) and fractional pump flow (FPF) were also calculated.RESULTS: SP increased lymphatic CF, TI and FPF, and the effects were enhanced with the increase in the concentration of SP. The CF, TI and FPF of 2 h- and 3 h- shocked lymphatics were elevated to/over the value of baseline levels before the experiment by SP at the concentration starting from 3×10^-8 mol/L. At the same concentration of SP, the CA of lymphatics showed no significant statistical difference among the groups. However, with the increase in SP concentration, the lymphatic CA had a downward trend in all groups.CONCLUSION: SP enhances the pump function of lymphatics not only under physiological condition, but also in shock during different stages.     

Effect of hypertonic saline solution on the geometry of erythrocyte in rats subjected to hemorrhagic shock

AIM:To study the effect of hypertonic saline solution on the geometry of erythrocyte in hemorrhage-shocked rats,using micropippette aspiration technique. METHODS:Wistar rats were randomized into three groups of 0.9% NaCl(NS),7.5% NaCl(HS) and 5% NaCl-3.5% NaAc(HSA). The animals were bled to reach a mean arterial pressure of 5.3 kPa in 10 minutes and maintained in shock for 90 minutes. 4 mL/kg NS,HS or HSA was given intravenously and respectively in 5 minutes following hemorrhagic shock. The blood was collected to determine the geometry of erythrocyte at baseline,after shock and treatment. RESULTS:There were no significant difference in surface area/volume ratio and spherical index between baseline and shock.The diameter of erythrocyte were decreased obviously following hemorrhagic shock. The surface area/volume ratio was significantly lower in HS group than that in NS and HSA group after treatment. HS and HSA raised spherical index of erythrocyte remarkbly compared with NS.Otherwise,the spherical index of erythrocyte was reduced significantly in HSA group compared to HS group. Both HS and HSA decreased diameter of erythrocyte notably compared with NS. CONCLUSION:Both HS and HSA have deleterious effects on the geometry of erythrocyte in rats subjected to hemorrhagic shock.Whereas, HSA could decrease the side effect compared with HS.     

Role of myosin-light-chain kinase in biphasic contractile activity of lymphatics isolated from hemorrhagic shock rats

AIM: To elucidate the mechanism by which myosin-light-chain kinase (MLCK) modulates the biphasic contractile activity of lymphatics isolated from the rats subject to hemorrhagic shock (HS). METHODS: Male Wistar rats were randomiz to control group and HS group. In HS group, the rats were subject to HS and then further divided into HS 0 h, 0.5 h, 1 h, 2 h and 3 h subgroups. Thoracic ducts of control and shock rats were isolated and used to determine the protein levels of phosphorylated MLCK (p-MLCK). In addition, thoracic ducts obtained from control, 0.5 h- and 2 h-shocked rats were used to observe the contractile properties of lymphatics by a pressure myograph in vitro . Lymphatic rings were prepared and incubated with ML-7 (a specific inhibitor of MLCK) or substance P (SP, an agonist of MLCK). During the experiment, the contractile frequency (CF), end-diastolic diameter, end-systolic diameter and passive diameter in Ca²⁺-free PSS buffer were measured and used to calculate the lymphatic tonic index (TI), contractile amplitude (CA) and fractional pump flow (FPF) as the indexes of lymphatic contraction activity. RESULTS: The levels of p-MLCK in lymphatics in 0 h- and 0.5 h-shocked rats were significantly increased compared with the control rats, and it was gradually decreased with the development of shock. The values of CF, TI and FPF in 0.5 h-shocked lymphatics were significantly increased at transmural pressure of 1, 3 and 5 cmH₂O compared with those in control group, and significantly blunted by ML-7. SP obviously increased the suppressive effects induced by ML-7 and restored the values of CF, TI and FPF to the levels of HS 0.5 h group. CF, TI and FPF in 2 h-shocked lymphatics significantly declined under different transmural pressure as compared with those in control group, and significantly elevated by SP. Similarly, ML-7 depressed the effects of SP. No significant difference was found in CA between 0.5 h- and 2 h-shocked lymphatics. SP decreased the CA of lymphatics obtained from 2 h-shocked rats and this effect was suppressed by ML-7. However, both agents had no effects on CA of 0.5 h-shocked lymphatics. CONCLUSION: MLCK, as an essential enzyme that influences the contraction of lymphatic smooth muscle cells, involves in the modulation of biphasic changes of lymphatic contractile activity during the process of HS.     

Protective effect of mesenteric lymph duct ligation on organ functions in MODS rats

AIM: To study the protective effect of mesenteric lymph duct ligation on the functions of liver, kidney and heart, and morphology in multiple organ dysfunction syndrome (MODS) rats subjected to two-hit. METHODS: Male Wistar rats were divided into three groups: the mesenteric lymph duct ligation group, the non-ligation group and sham group. The MODS model of two-hit was established by bleeding and LPS administration in both ligation group and non-ligation group. After 24 h, all rats were cannulated to facilitate blood withdrawal for serum sample, then all rats were killed and organs including kidney, liver, lung and heart were collected for making microscopic sections. The biochemical indexes of hepatic and renal functions and myocardial enzyme in serum were determined before and after experiment. RESULTS: After two-hit, the serum contents of AST, ALT, TBA, BUN, Cr and LDH-1 in both non-ligation group and ligation group, and UA content in non-ligation group were obviously increased than those in pre-experiment and sham group (P<0.01). The serum contents of ALT, TBA, BUN, Cr and UA in ligation group were obviously lower than those in non-ligation group (P<0.01, P<0.05). The tissue structures in kidney, lung, liver and heart in sham group were normal. However, congestion, degeneration and necrosis were found in organs in non-ligation group, and only mild lesions were found in ligation group. CONCLUSION: The results demonstrate that the ligation of mesenteric lymph duct improves the disturbance of organ function and pathomorphological variation in MODS rats subjected to two-hit by hemorrhage and LPS. The lymphatic mechanism in MODS should be worth to pay attention in further study.