| Shock is a severe syndrome related to a problem with the body's circulatory hypoperfusional state which may result in multiple organ dysfunction and a high mortality rate.Venous access and fluid therapy should still be considered to be essential elements of pre-hospital advanced life support(ALS) in the critical obstetric hemorrhagic patient.However,in recent years this treatment has been challenged,and clinical and experimental models have shown possible deleterious effects of fluid administration before hemorrhage control.Clinical practice guidelines and some national guidelines are now moving away from this traditional aggressive fluid resuscitation strategy in recommendation of a more judicious use of fluids. Permissive hypotensive resuscitation allows the restoration of blood circulation with minimal fluid requirements and modest increase in blood pressure,thus reducing the risk of additional blood loss due to continued bleeding or re-bleeding.The International Conference held in Paris,on April,27-28,2006 recommended a target blood pressure during initial shock resuscitation for uncontrolled hemorrhage due to trauma:MAP of 40mmHg until bleeding is surgically controlled.This theory in the clinical treatment of hemorrhagic shock in trauma has made good effect on reducing the complications of shock and improving patient outcome.Acute postpartum hemorrhage is the leading worldwide cause of maternal mortality,such deaths being usually related to the development of hemorrhagic shock. Due to special features of maternal hemodynamics,such as increased blood volume and hypercoagulable,whether hypotensive resuscitation could be applied to maternal hemorrhagic shock remains unknown.So far,there has been no study on this issue.Previous experimental studies we had conducted in an uncontrolled hemorrhagic shock of pregnant rabbits since 2000 year demonstrated the effectiveness of small-volume limited fluid resuscitation to achieve hemodynamic stability,reduce re-bleeding and improve survival time.The study was consistent with the known findings in animal models or patients with penetrating trauma.However,there are many controversy surrounds optimal resuscitative fluids,volume and physiological goals,particularly in prehospital setting before definitive hemorrhage control.The present study is designed as a prospective,randomized laboratory experiment to investigate the impact of fluid resuscitation on hemodynamic parameters,microcirculation perfusion,energy metabolizability,ischemia reperfusion injury and long-term survival rate in a clinically relevant model of uncontrolled hemorrhagic shock in pregnancy.To evaluate the pathological and physiological mechanism of hemorrhagic shock in pregnancy and the efficacy of hypotensive resuscitation strategies on uncontrolled hemorrhagic shock in pregnancy.provide a reliable basis for further applications in the clinical research,so as to improve pre-hospital emergency treatment,and reduce maternal mortality rates.Chapter 1 Effect of fluid types of limited resuscitation on microcirculation in an uncontrolled hemorrhagic shock of pregnant rabbitsOBJECTIVEThe observations indicate that the restoration of systemic conditions do not necessarily lead to the restoration of microvascular function.Capillary no-reflow post-treatment lead to decrease of Functional capillary density(FCD),which is a crucial factor for the different outcome of hemorrhagic shock.The purpose of this study is to determine the effects of different fluid resuscitation strategies on the rabbit chamer microcirculation,hemodynamic parameters,metabolism and long-term survival rate in a clinically relevant model of uncontrolled hemorrhagic shock in pregnancy and to evaluate the effect of hypotensive resuscitation strategies on microvascular perfusion in uncontrolled hemorrhagic shock.METHODS1,Tablish pregnant rabbit ear chamer model with hemorrhagic shock:After anesthesia,Skin Preparation,carotid artery catheterization,rabbit ear chamer Preparation,open abdominal incision along the lower abdomen revealed womb, production pregnant rabbit model with hemorrhagic shock.The experiment consisted of four phases,basic phase(0min),shock phase(0-30min),prehospital phase (30-90min),and hospital phase(90-180min).In the shock phase,a 3-4mm arterial vessel in mesometrium of uterus was transected,and enclosed with aseptic sponges (for blood loss calculation).Rabbits were hemorrhaged by blood withdrawal to mean arterial pressure(MAP) of 40-45mmHg at an average rate of 2ml.kg-1min-1 within 15min(blood was aspirated into a sterile heparinized syringe).After this initial hemorrhage,blood was withdrawn or reinfused as needed to maintain this level for 15min.In the prehospital phase,Then animals were underwent resuscitation protocol assigned previously.In hospital phase,bleeding was controlled by surgical intervention,therefore blood vessel was ligated,and animals were reinfused with fluid and shed blood to MAP of 80mmHg and maintained that level for 90min.At T180min,the cannulas were then removed from the animals after ligation of the femorary artery and vein and the incisions were closed.The animals were placed in a caged with food and water and observed for survival for 7 days.2,Experimental protocol:We categorized thirty New Zealand Rabbits,who were in their middle or late pregnancy stages(15-25 days),into three groups(n=10).The NS, NH,HHH Groups represent three cases with which we injected various treatment liquids thirty minutes after the tested rabbits entered the shocking stage.The tested rabbits in the NS Group were rapidly injected with a large quantity of normal sodium (NS) and lactated Ringer's(LR) in order to maintain MAP at the approximately 80 mmHg level.The tested rabbits in the NH Group were slowly injected with a small quantity of NS(4ml/KG) and LR in order to maintain MAP at the approximately 60 mmHg level.The HHH Group were slowly injected with a small quantity of HHES(a mixure of 10%hydroxyethyl starch and 7.5%hyperisotonic sodium,4ml/KG)and NS,LR in order to maintain MAP at the approximately 60mmHg level,All rabbits in each group accepted standard surgical stop-bleeding and injecting blood/liquid treatments in hospital stage.3,Blood samples for BE,PH,PCO2,PO2 and HCT were taken at 0min,30min, 90min,180min after the start of the experiment.Peripheral microcirculation and MAP was recorded continuously.pH,BE PCO2,PO2were determined with a Medica Easy Blood Gas Analyzer(Medica Corporaton,U.S.A.).Blood HCT was measured by Micro Hematocrit Centrifuge(Zhengji instrument Inc.Jintan,China.).Functional capillary density(FCD) was analyzed online by counting the number(percentage) of capillaries segments that have RBC transit of at least one RBC in a 30-sec period, was analyzed from four to six different video-recorded microscopic fields exhibiting four to six different capillaries each.At the end of the experiment,and free intraperitoneal blood was collected on preweighed pieces of cotton,infused volume was recorded,the laparotomy incision was stitched.Pregnant rabbits were used to observe the effects of different resuscitation strategies on a 7-day period and survival rated was record.RESULTS1.Changes of blood pH,BE,PO2,PCO2 in pregnant rabbits among different groups: Shock caused a significant increase of arterial PO2 and decrease of PCO2,pH,and base excess.At the end of the resuscitation,all the groups tended to recover from the hyperventilation except NS groups.There was no significant difference among the groups(P＞0.05 ).pH,BE value tended to increase at the end of hospital resucitation phase in all the groups but not reach to base period,At the end of the resuscitation, The pH,BE value was significantly higher in NS groups than the other groups(P＜0.05).2.Comparison of arterial and venule diameter among different groups:Hemorrhagic shock resulted in a similar and significant constriction of arterial diameter(0.63±0.54 of baseline) and venule diameter(0.77±0.67 of baseline).After resuscitation,arteriole diameter in all the groups were increased and reached significant difference among three groups(F=103.932,P=0.000 ),from the highest to lowest value is HHH(0.92±0.04%),NH(0.85±0.07%) and NS(0.71±0.01%) respectively.Although all groups tended to recover from venule constriction after resucitation to 0.86±0.68 of the baseline values;these changes,however,remained significantly lower than baseline values and did not reach significant difference among groups.3.Comparison of FCD among different groups:Hemorrhagic shock also caused a significant reduction of FCD in all animals with values being lowered to 42.57±5.5 of baseline.Although resuscitation increased FCD in all animals relative to the shock period,normal values could not restablished.At the end of hospital resuscitation,FCD levels were NS(59.70±4.81%),NH(70.30±5.62%),HHH(91.70±4.69%) relative to baseline,respectively,with significant differences between groups(F=103.932, P=0.000),All the groups remained at significantly smaller values than the baseline. FCD at the end of the hospital resuscitation were significantly related with survival time(r=0.505,p=0.004 ).4.Comparison of blood loss and infused volume among different groups:hypotensive resuscitation provided superior outcomes than aggressive resuscitation based on fluid balance,blood loss or mortality,HCT at the end of the hospital resuscitation were significantly related with survival time(r=0.379,p=0.03 ).CONCLUSIONWe find that in uncontrolled hemorrhagic shock model of pregnant rabbits,both resuscitation velocity and fluid ingredient had different effect on microcirculation and metabolism.Compared with aggressive fluid resuscitation,hypotensive resuscitation reduce constriction of arterial and venule diameter,increase FCD and microcirculation perfusion,improved long-term survival.Hypertonic salt and colloid had proven efficacy to ameliorate microcirculation without improving survival rate.The composition of the ideal fluid need be lucubrated. CHAPTER 2 Effect of fluid types of limited resuscitation on protective immunity in lungs in an uncontrolled hemorrhagic Shock of pregnant rabbitsOBJECTIVEThe inflammatory response after shock is believed to be a key step leading to host tissue damage,and it is mediated largely by the activation of neutrophils.This inflammatory response induce the the activation of neutrophils and other phagocytes,resulting in serious tissue damage and,ultimately,end organ failure.The purpose of this study was to determine the effects of different fluid resuscitation strategies on the blood PAF,level MPO activity extracted from lung tissue and mitochondrion ultrastructure in lung in a clinically relevant model of uncontrolled hemorrhagic shock in pregnancy.To discuss the impact of limited fluid resuscitation on the occurance of systemic inflammatory response and organ function in pregnant rabbit with hemorrhagic shock.METHODS1,Tablish pregnant rabbit model with hemorrhagic shock:After anesthesia,Skin Preparation,carotid artery catheterization,open abdominal incision along the lower abdomen revealed womb,production pregnant rabbit model with hemorrhagic shock.The experiment consisted of four phases,basic stage(0min),shock phase (0-30min),prehospital phase(30-90min),and hospital phase(90-180min).In the shock phase,a 3-4mm arterial vessel in mesometrium of uterus was transected,and enclosed with aseptic sponges(for blood loss calculation),rabbits were hemorrhaged by blood withdrawal to mean arterial pressure(MAP) of 40-45mmHg at an average rate of 2ml.kg-1min-1 within 15min(blood was aspirated into a sterile heparinized syringe).After this initial hemorrhage,blood was withdrawn or reinfused as needed to maintain this level for 15min.In the prehospital phase,Then animals were underwent resuscitation protocol assigned previously.In hospital phase,bleeding was controlled by surgical intervention,therefore blood vessel was ligated,and animals were reinfused with fluid and shed blood to MAP of 80mmHg and maintained that level for 90min.At T180min,the cannulas were then removed from the animals after ligation of the femorary artery and vein and the incisions were closed.The survivors were sacrificed,organs were harvested for determination.2,Experimental protocol:We categorized eighteen New Zealand Rabbits,who were in their middle or late pregnancy stages(15-25 days),into three groups(n=10).The NS,NH,HHH Groups represent three cases with which we injected various treatment liquids thirty minutes after the tested rabbits entered the shocking stage.The tested rabbits in the NS Group were rapidly injected with a large quantity of normal sodium (NS) and lactated Ringer's(LR) in order to maintain MAP at the approximately 80 mmHg level.The tested rabbits in the NH Group were slowly injected with a small quantity of NS(4ml/KG) and LR in order to maintain MAP at the approximately 60 mmHg level.The HHH Group were slowly injected with a small quantity of HHES(a mixure of 10%hydroxyethyl starch and 7.5%hyperisotonic sodium,4ml/KG)and NS,LR in order to maintain MAP at the approximately 60mmHg level,All rabbits in each group accepted standard surgical stop-bleeding and injecting blood/liquid treatments in hospital stage.3,Blood samples for PAF were taken at baseline,shock and resuscitation.At the end of the experiment,survivors were sacrificed,lungs were harvested for determination of MPO activity.The mitochondrion parameters of morphometry were examined by electron microscopy.The two reagents of PAF and MPO were purchased in the United States ADE Corporation,Nanjing jiancheng Corporation respectively.RESULTS1.Comparison of MPO activity in lungs among different groups:Tissue MPO activity in lungs were significantly lower in the HHH(1.86±0.25 U/L) and NH (2.47±0.23U/L) group than NS(3.05±0.34U/L) group(F=27.067,P=0.000).2.Comparison of plasma PAF among different groups:plasma PAF concentrations increased in all the groups at the end of the shock phase and continued to increase until the end of hospital phase.PAF concentrations in the end of hospital phase from highest to lowest is NS(4.41±0.12pg/ml),NH(3.97±0.13pg/ml),HHH(3.57±0.15 pg/ml),and there were significantly difference among the groups(F=58.562, P=0.000).Total blood plasma PAF concentrations in NH group were significantly less than NS group and larger than HHH group.3.Assessment of lung damage:At the end of the experiment,The cell structure in NS group were destyoyed seriously compared with that in NH and HHH group. Manifestation of lung tissue under electron microscope:TypeⅡcells of alveoli swelled and the tiny villus reduced,mitochondrial crista disappered,expanded,the lamellar body was reduced,with some empty cell.CONCLUSIONWe find that in uncontrolled hemorrhagic shock model of pregnant rabbits, hypotensive resuscitation eased the lung inflammation damages lead by PAF incease and neutrophil collection more than aggressive fluid resuscitation.The hyperosmotic crystalloid and hyperonoctic colloid resuscitation could clearly limit the same damages.CHAPTER 3 Effect of fluid types of limited resuscitation on ATPase activity in an uncontrolled hemorrhagic shock of pregnant rabbitsOBJECTIVEThe basic mechanisms that underlie the development of cell.tissue and organ damage in shock syndromes depend in part on the duration and the severity of the injury.The hypoxic cell is compromised in multiple ways.Cell energy metabolism is switched from aerobic to anaerobic glycolysis.This leads to the cellular accumulation of lactate,hydrogenion and inorganic phosphates.Levels of ATP in the cell are decreased due to diminished synthesis,continued consumption and the actions of ATPase.The purpose of this study was to determine the effects of different fluid resuscitation strategies on ATPase activity in various tissue,on the blood LA,Na+,K+ level in a clinically relevant model of uncontrolled hemorrhagic shock in pregnancy.To discuss the impact of limited fluid resuscitation on the energy metabolizability in pregnant rabbit with hemorrhagic shock. METHODS1,Experimental grouping and methods as same as chapter 2.2,Blood samples for lactate,Na+,K+ were taken at baseline,shock and resuscitation. At the end of the experiment,survivors were sacrificed,and the heart, liver,lung,kidey,small intestine,and skeletal muscle were harvested for determination of Na+-K+-ATPase and Ca2+- Mg2+-ATPase activity.Coming 634 Electrolyte Analyzer measures the concentration of Na+,K+;the reagent of ATPase were purchased in Nanjing jiancheng Corporation.RESULTS1,Comparison of plasma ATPase activity among different groups:At the end of the experiment,Na+-K+-ATPase activity was more higher than Ca2+- Mg2+-ATPase in various tissue.Animals assigned to hypotensive fluid resuscitation therapy had a significantly lower change in ATPase activity in skeletal muscle,heart,liver,kidney than the animals assigned to aggressive or delayed fluid resuscitation therapy.2,Comparison of plasma concentration of Na+,K+ among different groups: Animals assigned to hypotensive fluid resuscitation therapy had a significantly lower lactate concentration,a lower K+ concentration than the animals assigned to aggressive fluid resuscitation therapy.Serum sodium concentrations were only transiently elevated in prehospital phase in the HHH group,versus the other group, and tended to return to baseline levels at hospital phase.The plasma lactate concentration increased,with increasing Na+-K+ -ATPase activity in skeletal muscle at the end of the hospital resuscitation(pearson correlation coefficient:r=0.895, P=0.000).CONCLUSIONWe find that shock results in interruption of energy metabolism in uncontrolled hemorrhagic shock model of pregnant rabbits.Its impacts on various organs are different but are more conspicuous on the changes in ATPase activity in skeletal muscle and heart.Compared with aggressive fluid resuscitation,hypotensive resuscitation relatively stablized energy metabolosm and reduced the damages to cell membrane and cellular organelle.CHAPTER 4 Effect of fluid types of limited resuscitation on ischemia- reperfusion injury on various organs in an uncontrolled hemorrhagic shock of pregnant rabbitsOBJECTIVEThe process of adequate resuscitation after hemorrhagic shock is,to some extent, similar to that of the ischemia and reperfusion,which has an important role in tissue damage following hemorrhagic shock.Presently,there is no report regarding ischemiareperfusion injury in an uncontrolled hemorrhagic shock model,and the effect of early limited resuscitation on tissue damage is unknown.The purpose of this study was to determine the effect of three fluid resuscitation strategies on the SOD and MDA activity extracted from various tissue in a model of uncontrolled hemorrhagic shock in pregnancy rabbit.we explore the effect of different fluid resuscitation on the tissue oxidation injury and subsequent treatment during prehospital and hospital phases.METHODS1,Experimental grouping and methods as same as chapter 2.2,At the end of the experiment,survivors were sacrificed,and the heart, liver,lung,kidey,small intestine.and skeletal muscle were harvested for determination of malondialdehyde content(MDA) and total antioxidative capacity(SOD).the reagent of MDA,SOD were purchased in Nanjing jiancheng Corporation.RESULTS1,The chang of total antioxidative capacity and malondialdehyde content after hemorrhagic shock:The ischemia and reperfusion injury in uncontrolled hemorrhagic shock resulted in the decline of total antioxidative capacity and the increase of malondialdehyde content;The ischemia and reperfusion injury is a generalized reaction involving various organs.the damage of lung and intestine tissue is more serious than the others,of which lung tissue is the most serious,whereas the heart and skeletal muscle is more lighter. 2,Comparison of plasma total antioxidative capacity and malondialdehyde content among different groups:The malondialdehyde content in HHH and NH Group was significantly lower than that of NS Group,whereas The total antioxidative capacity was significantly higher in HHH and NH Group than that of NS Group in the tissue of liver,lung,kidey,and small intestine(lung:F=9.285,P=0.002;liver:F=12.346, P=0.001;kidey:F=11.039,P=0.001;small intestine:F=25.006,P=0.000);The malondialdehyde content in HHH Group was significantly lower than that of NH Group,whereas The total antioxidative capacity was significantly higher in HHH than that of NH Group(lung:F=11.155,p=0.001;liver:F=21.549,p=0.000;kidey: F=10.504,p=0.001;small intestine:F=12.174,p=0.001).CONCLUSIONThe limited resuscitation relief in pre-hospital phase had benefit for subsequent treatment in the hospital phase in terms of alleviated tissue damage and improved survivorship.The ischemic injury of various organs showed significant correlation with the others.These results suggested that the blockage of damage in initial and major organs might play an important role in the remedy of hemorrhagic shock.SUMMURY1,Compared with aggressive fluid resuscitation,hypotensive resuscitation reduces constriction of arterial and venule diameter,increases FCD and microcirculation perfusion and improvs long-term survival.Hypertonic salt and colloid have proven efficacy to ameliorate microcirculation without improving survival rate.The composition of the ideal fluid need be lucubrated.2,We find that in uncontrolled hemorrhagic shock model of pregnant rabbits, hypotensive resuscitation eased the lung inflammation damages lead by PAF incease and neutrophil collection more than aggressive fluid resuscitation.The hyperosmotic crystalloid and hyperonoctic colloid resuscitation could clearly limit the same damages.3,We find that shock results in interruption of energy metabolism in uncontrolled hemorrhagic shock model of pregnant rabbits.Its impacts on various organs are different but are more conspicuous on the changes in ATPase activity in skeletal muscle and heart.Compared with aggressive fluid resuscitation,hypotensive resuscitation relatively stablized energy metabolosm and reduced the damages to cell Membrane and cellular organelle.4,The limited resuscitation relief in pre-hospital phase had benefit for subsequent treatment in the hospital phase in terms of alleviated tissue damage and improved survivorship.The ischemic injury of various organs showed significant correlation with the others,these results suggested that the blockage of damage in initial and major organs might play an important role in the remedy of hemorrhagic shock.In summary,we elucidate the efficacy and mechanism of limited volume resuscitation in pregnant rabbits of uncontrolled hemorrhagic shock.
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