| Objectives:Hemorrhagic shock(HS) might cause systemic inflammatory response syndrome (SIRS). It would certainly result in multiple organ dysfunction syndrome (MODS) if the development of SIRS was out of control. SIRS in lungs was manifested by acute lung injury (ALI). Obviously, the process, SIRS→ALI→ARDS→MODS, reflected the dormino effects. Therefore, the determination the pathogenesis of ALI/ARD, and the early prevention and treatment for ALI/ARD, were significant for reducing mortality rate and impoving the prognosis of HS.Intestinal bacteriemia and endotoxemia induced by HS, was an important causative factor of ALI/ARDS. And the main mechanisms of ALI/ARDS after HS, were the resulting over activation of polymorphonuclear neutrophils (PMNs), the incontrollable inflammation in lungs, and the necrosis or apoptosis of pulmonary vascular endothelial cells. During the process of ALI/ARDS, the inflammation response resulted in the increased permeability of lungs. The endogenous ARDS usually appeared on pulmonary microvascular endothelium, and the exogenous ARDS on alveolar epithelium. The main reasons that pulmonary microvascular endothelium permeability increases were the release of inflammatory mediator, the interaction between polymorphonuclear neutrophils and endothelial cells, and the changes of cytoskeletons.A prior consideration should be taken, that was to improve the pathology state and survival rate on the premise of regaining the hemodynamics, when chosing resuscitation fluid after a hemorrhagic shock, especially in the shortage of blood. Hydroxyl starch (HES) was the most common plasma volume expander on clinical application, whose expansion capability and maintenance was determined by its concentration, relative molecular mass, and the degree or patterns of gram molecule substitution. 6%HES130/0.4(hydroxyethyl starch 130/0.4, Voluven)was a new generation of HES, with a gram molecular weight of 130,000 dalton, substitution degree of 0.4, and C2/C6 hydroxyethylated rate of 9:1. Compared with other HES fluids and daxtran, it had the same effect of volume expansion, but less side effects. Besides a good expansion effect, the 6%HES130/0.4 could also reduce the leucocytes migration, the inflammatory reaction and endotoxin-induced ALI in rats. But it had no fixed conclusion about the effects and mechanisms for the damage of non-infectious pulmonary inflammatory.This study was carried out with experimental surgery technique. Models with hemorrhagic shock were made in rats by blood loss from carotid. Then invasive monitoring of arterial pressure method was used to resuscitate rats in HS with different shock-time duration, and estabilished the SD rat ALI models. Based on these, HES130/0.4 was used for early intervention. To observe the changes of pulmonary function, pathomorphology and ultra-microstructure, TNF-α,IL-10 in lung tissue homogenates and CD11b\CD18 of pulmonary polymorphonuclear neutrophils were made; to study the effects or the mechanism of pulmonary vascular endothelial cell and polymorphonuclear neutrophils in the pocess of ALI induced by HS fluid resuscitation, and the effects and mechanisms of HES130/0.4 on the early intervention with ALI induced by HS fluid resuscitation, several detection technologies were used here, such as fully auto blood gas analysis, pathomorphological observation, transmission electronic microscope, flow cytometry, enzyme-linked immunosorbent assay, and so on. On the basis of animal experiment, the effects and mechanism of HES130/0.4 on the damage of pulmonary microvascular endothelial cells had been futher observed. So, new treatment strategies and theories were provided for early intervation with ALI induced by hemorrhagic shock and fluid resuscitation.Methods: Part I: Establishing models of acute lung injury induced by hemorrhagic shock and resuscitation in ratsFourty-eight SD rats were divided into 6 groups (45min hemorrhagic group,60min hemorrhagic group,and 90min hemorrhagic group and their corresponding control groups) by means of random number table. The control operation group was only anesthetized and dealt with venipuncture indwelling catheter,without removal of blood or fluid resuscitation. Different shock duration groups were divided into group H45, group H60, group H90 by time. The models of hemorrhagic shock in rats established with the method of blood in carotid atery were resuscitated by infusing Ringer's lactate of 3 times the maximum volume of the removed blood in femoral veins after shock. Then, the hemorrhagic level of different groups were calculated; MAP at different time points were recorded; blood gas analysis were taken before shock, before fluid resuscitation and during the 3 hours resuscitation separately, and the PO2/FiO2 ratios were calculated. These animals were killed quickly by the method of removal blood in carotid artery after 3 hours resuscitation. Then the lung congestion, edema, hemorrhage were generally observed in each group. The pulmonary pathological changes at light microscope observed and the lung pathological score were calculated in the different groups. And the ultrastructure in lung tissue was observed by transmission electron microscope (TEM). The brochoalveolar lavage fluid (BALF) protein concentration and the wet/dry weight (W/D) ratio of lung were determined.Part II: Effect of 6% HES 130/0.4 resuscitation on lung TNF-α, IL-1βand IL-10 in hemorrhagic shock ratsTwenty-four adult male SD rats were divided into 4 groups: control operation group (group control) , Ringer's lactate group (group RS), resuscitation group of 33ml/kg (group H1), resuscitation group of 50ml/kg (group H2) by the means of random number table. The rats in control group was anesthetized and dealt with only venipuncture,without blood removal. After shock, Ringer's solution of 3 times the maximum volume of the removed blood was infused in femoral veins to make resuscitation in group RS. 33ml/kg of 6% HES130/0.4 were infused to make resuscitation in group H1 and 50ml/kg in H2 group. To ensure the same effect of theoretical resuscitation, the Ringer's solution equalled 3 times the maximum volume of the removed blood minus corresponding amount of hydroxyethyl starch in H1and H2 were infused.The largest hemorrhagic level of different shock groups were calculated; MAP at different time points were recorded; Blood gas analysis were made before shock (T0), before resuscitation (T1), after 2 hrs (T4) and 3 hrs (T5) of resuscitation; PO2/FiO2 ratio were calculated. Rats were killed after 3 hrs of resuscitation. BALF protein concentration and the lung wet/dry weight (W/D) ratios were determined. The concentrations of tumor necrosis factor-α(TNF-α), interleukin-1 (IL-1β) and interleukin-10 (IL-10) in lung issue were determined. Generally the lung congestion, edema, hemorrhage in each group were observed. The pulmonary pathological changes were observed at light microscope and the lung pathological score were calculated in the different groups. The ultrastructural organization were observed in lung tissue by TEM.Part III: Effect of 6% HES 130/0.4 resuscitation on lung MDA, SOD and MPO in hemorrhagic shock ratsRats was selected and classified as in the second part.The rats were killed at 3h after fluid resuscitation in shock groups, and at the same time in to obtain the lower lobe lung issue in right lung. And the amount of MDA, activity of SOD and MPO were determined.Part IV: Effect of 6% HES 130/0.4 resuscitation on the expression of CD11b and CD18 in arterial PMNs in hemorrhagic shock ratsRats'selection, group and establishing models of hemorrhagic shock were the same as described in second part. To reduce the differences in different groups during the test with flow cytometry (FCM), this part of the experimental rats were divided into batches, each batches of four, divided into control operation group (group S),Ringer's lactate group (group RS) , resuscitation group of 33ml/kg (group H1) , resuscitation group of 50ml/kg (group H2) Blood gas analysis were made before shock (T0), before resuscitation (T1), 2 hours (T4) and 3 hours (T5) after resuscitation separately; Flow Cytometry (FCM) were detected for the expression level of polymorphonuclear neutrophils (PMNs) of CD11b and CD18 in artery blood.Part V: Exprimental study of the effect of 6% HES 130/0.4 on the apoptosis rate of injuried pulmonary microvascular endothelial cell induced by LPS1. Modified tissue block pasted culture method was used in primary culture of pulmonary microvascular endothelial cell (PMVECs). The tissue block were inverted under the phase contrast microscope and the cellular morphology were observed. The cellular morphology and ultrastructure were observed with scanning electron microscope (SEM) and TEM, and the cells were identified. 2-3 generation of cells cultured were used in next experiments.2. Suspension concentration of the first part of PMVECs was adjusted to 1×109/ml. 1ml of cell suspension was added into each hole of a 24 holes'cell culture plate. A total of 15 holes were randomly divided into 3 groups: control group (group C),LPS treatment group (group L) and HES intervention group (group H). Group L was added with 1μg/ml LPS and other fluid PBS with the same volume. H group was added with 1μg/ml LPS and other fluid PBS with the same volume and HES130/0.4 30mg/ml. Group C was added with the fluid PBS with the same volume. The cell culture plates were put into cell incubators, removed after 3 hours and the cells were collected. The cellular morphology and ultrastructure were observed by SEM and TEM. Apoptosis rate and cell proliferation of PMVECs were detected by flow cytometer. Results:Part I: Establishing models of acute lung injury induced by hemorrhagic shock and resuscitation in rats1. Each group of rats were alive till the experiments. It was meaningless in statistics in the groups of shock about the blood loss level and the interblock diference of MAP. 2. Compared with the value in group control, PaO2/FiO2 rised at time point T1, PaCO2 were decreased (P<0.05) in three shock groups; in group H45 and H90, PaO2/FiO2 decreased at time point T4 (P<0.05). Comapared with the value in group H45 and H60, PaO2/FiO2 rised at time point T5, and PaCO2 decreased (P<0.0) in group H90. Compared with the value in group H60, PaO2/FiO2 decreased at time point T5, and PH rised at time point T4 and T5 in group H90 (P<0.05).3.1 The general observing results of lung tissue: no obvious changes were observed in the group control. Ischemia, congestion and oedema in various degrees could be found in each group of shock rats, espessially in group H90.3.2 The structure of alveolus was not found obvious abnormity. In the groups H45 and H60, the lung interstitial substance was broadened, and inflammatory cell infiltration could be found. In group H60, inflammatory exudation and lammatory infiltration in small bronchus could be found. Hematid and inflammatory cell in the intracavity of alveolus could be found, the broadened lung interstitium, inflammatory cell infiltration, expansion and congestion interstitium minute vessel could be found in group H90. Compared with the values in group control and H45, group H60 and H90, pathology grade of lung tissue rised (P<0.05). Compared with the value in group H60, pathology grade of lung tissue in H90 rised (P<0.05).3.3 In group control, there was no obvious abnormity found in ultrastructure of lung tissue. In group H45, alveolitoidⅡcell was basically nomal, lamellated granule was nomal, but mitochodrial membrane was impairment.In group H60, the interstistial substance of lung was broaden, inflammatory cell intitration, inflammatory exudation and lammatory infiltration cound be found in small bronchus. In group H90, hematid and inflammatory cell in the intracavity of alveolus, the broadened interstitial substance of lung, inflammatory cell infiltration, expansion and congestion of interstritial substance of minute vessel could be found.3.4 Compared with the value in group control, viscosity albuminose of BALF and W/D of lung rised in each shock group (P<0.05 or 0.01); Compared with the value in group H45 and H60, lung viscosity albuminose of BALF and W/D rised (P<0.05or 0.01) in group H90.Part II: Effect of 6% HES 130/0.4 resuscitation on lung TNF-α, IL-1βand IL-10 in hemorrhagic shock rats1. There was no difference statisticly of MAP at different time points between fluid resuscitation groups.2. Compared with the value in group control, PaO2/FiO2 increased and PaCO2 decreased (P<0.05) at time point T1 in group H1, H2 and RS. In group RS, PaO2/FiO2 increased at time point T4 and T5. PaO2/FiO2 in group H2 increased at time point T5 (P<0.05). Compared with the value in group RS,, PaO2/FiO2 increased at time point T4 and T5 in group H1. PaO2/FiO2 increased at time point T4 in group H2. In the group H1 and H2, PaCO2 increaesd at time point T4 (P<0.05). Compared with the value in group H1, PaO2/FiO2 decreased at time point T5 (P<0.05) in group H2.3. Compared with the value in group control, IL-10 and protein in BALF and lung W/D increased in group RS, H1 and H2 (P<0.05); compared with the value in group RS, the concentration of TNF-α, IL-1β, the protein in BALF and lung W/D in group H1 and H2 decreased, while the concentration of IL-10 increased (P<0.05).4.1 The observed results of lung tissue: there was no obviously change in group control; Ischemia, congestion and edema in various degrees could be found in each fluid resucitation group, especially in group RS.4.2 The stucture of alveolus was nomal in group control. Hematid and inflammatory cell in the intracavity of alveolus, the broadened interstitial substance of lung, inflammatory cell infiltration, expansion an congestion of interstitial substance of minute vessel could be found in group RS. The broadened interstitial substitial of lung, inflammatory cell infiltration, and inflammatory exudationg and lammatory infiltration in interstitial substance of minute vessel could be found in group H1 and H2. The damage of lung tissue in group H1 and H2 were less than the group RS, the least damage of lung tissue was in group H1. Compared with the value in group control, the pathology grade of lung tissue increased in each of fluid resuscitation group (P<0.05). Compared with the value in group RS, the pathology grade of lung tissue was decreased in group H1 and H2 (P<0.05). Compared with the value in group H1, the lung tissue pathology grade increased in the group H2 (P<0.05).4.3 In group control, the ultrastructure of lung tissue was obviously nomal, while the ultrastructure of lung tissue was damaged in group RS. Basement membrane of blood vessel was reptured, epithelial microvillus was obviously reduced, rough surfaced endoplasmic reticulum was expanded, and the phenomenon of kermel integration could be found; Those in group H1 were basically nomal except slight structural modificationg of mitochondria; Perinucleat space was low-grade expanded, rough surfaced endoplasmic reticulum was low-grade expanded and there was phenomenon of degranlation in group H2.Part III: Effect of 6% HES 130/0.4 resuscitation on lung MDA, SOD and MPO in hemorrhagic shock rats1. Compared with the value in group control, the concentration of MDA increased in group RS, H1 and H2 (P<0.05); Compared with the value in group RS, the concentration of MDA decreased in group H1 and H2 (P <0.05); Compared with group H1, the concentration of MDA increased in group H2 (P <0.05);2. Compared with the value in group control, the concentration of SOD decreased in group RS, H1 and H2 (P<0.05); Compared with the value in group RS, the concentration of SOD increased in group H1 and H2 (P <0.05); Compared with the value in group H1, the concentration of SOD decreased in group H2 (P <0.05);3. Compared with the value in group control, the concentration of MPO increased in group RS, H1 and H2 (P<0.05); Compared with the value in group RS, the concentration of MPO decreased in group H1 and H2 (P <0.05); There was no difference statisticly of MPO beteween group H1 and H2 (P >0.05); Part IV: Effect of 6% HES 130/0.4 resuscitation on the expression of CD11b and CD18 in arterial PMNs in hemorrhagic shock rats1.There was no statistical significance of MAP at each time point between groups (P >0.05);2.Compared with the value in group control, PaO2 increased at T1, while PaCO2 decreased at T1~5 in every fluid resuscitation group (P<0.05); compared with the value at T0, PaO2 in every fluid resuscitation group at T1, group H1 at T1~5 and group H2 at T5 increased; PaCO2 in every fluid resuscitation group decreased at T1~5; except group H1, PH in every fluid resuscitation group was lower at T4, 5 (P<0.05);3.Compared with the value in group control, the expression of CD11b and CD18 in every fluid resuscitation group increased at T1, T4, T5 (P<0.05); compared with the value in group RS, the expression of CD11b and CD18 in both group H1 and H2 decreased at T4, T5 (P<0.05); compared with the value in group H1, the expression of CD11b and CD18 decreased at T4, T5 time in group H2 (P<0.05).Part V: Exprimental study of the effect of 6% HES 130/0.4 on the apoptosis rate of injuried pulmonary microvascular endothelial cell induced by LPS1 Under inverted microscope, cultured cells were cobblestone arrangement; microvilli and fenestrae on the surface of cultured cell could be observed by SEM. Protuberances on the surface of endothelial cells were observed; large and clear nucleolus, Golgi complex in the cytoplasm and rough endoplasmic reticulum as well as well developed smooth endoplasmic reticulum were observed by TEM.2 In group C, cell morphology with no significant changes were spindle or polygona by SEM; in both group L and group H, part of the cells became round shape, with decreased protrudings in cell edges but no apoptosis bodies were observed;3 Effects on the proliferation index and apoptosis rates of LPS-induced PMVECs HES130/0.4: compared with group C, PI of group L and group H was no statistical significance (P>0.05); compared with group L, PI of group H was no statistical significance (P>0.05); compared with group C, the apoptosis rate in group L and H increased (P>0.05); compared with group L, the apoptosis rate decreased in group H (P>0.05). Conclusions:Part I: Establishing models of acute lung injury induced by hemorrhagic shock and resuscitation in ratsAs hemorrhagic shock lasts for 45, 60 and 90 minutes while using Ringers solution to resuscitate for 2 or 3 hours may cause lung tissue changes functionally and morphologically, and the 90min is the most obvious one; and one reason of lung damage in hemorrhagic shock using fluid resuscitation during 45, 60 and 90 min is increasing of pulmonary capillary permeability, and the 90min was the most obvious one.Part II: Effect of 6% HES 130/0.4 resuscitation on lung TNF-α, IL-1βand IL-10 in hemorrhagic shock rats1. Lungs may have pathomorphological damage by using Ringers solution to resuscitation HS for 3 hours, and with 6%HES130/0.4 33ml/kg or 50ml/kg HES resuscitation can mitigate the pathomorphological change differently;2. The 33ml/kgHES make much more effort than the 50ml/kg one to mitigate the lung's pathomorphological change and caused by HS Part III: Effect of 6% HES 130/0.4 resuscitation on lung MDA, SOD and MPO in hemorrhagic shock ratsBy using 6%HES130/0.4 for HS fluid resuscitation can bring decreased the organic oxidation reaction conspicuously, and the 33ml/kg 6%HES130/0.4 is much more obviously than the 50ml/kg one.Part IV: Effect of 6% HES 130/0.4 resuscitation on the expression of CD11b and CD18 in arterial PMNs in hemorrhagic shock ratsThe fluid resuscitation and HS itself may induce the activation of neutrophils, 6%HES130/0.4 can reduce the activation of neutrophils; and 33ml/kg 6%HES130/0.4 is much more effective than the 50ml/kg one for HS fluid resuscitation. Part V: Exprimental study of the effect of 6% hydroxyethyl starch 130/0.4 on the apoptosis rate of injuried pulmonary microvascular endothelial cell induced by LPS1. When the damaged PMVECs induced by LPS are treated with HES130/0.4, the apoptosis rates increased.2. There is no connection between the cell cycle and apoptosis percentage of damaged PMVECs induced by LPS when treated with HES130/0.4.
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