Protective Effect Of Hypotensive Fluid Resuscitation For Intestinal Mucus Barrier In Pregnant Rabbit Model With Uncontrolled Hemorrhagic Shock

Massive hemorrhage is a common complication in Obstetrics. The increased tolerance for hemorrhage during pregnancy results in the delay of manifestations and failure of prompt management of hemorrhagic shock. Therefore, hemorrhagic shock remains one of the leading causes of maternal mortality. Traditionally resuscitation of the patients with hemorrhagic shock involves aggressive intravenous infusion. Now this notion is being challenged due to the increased mortality and morbidity. Therefore, hypotensive resuscitation may be preferable for patients with the potential of ongoing uncontrolled hemorrhage because it avoids the detrimental effects associated with early aggressive resuscitation while still maintaining the perfusion of critical organs. However, hypotensive resuscitation has been studied primarily in non-pregnant animals. Whether hypotensive resuscitation could be applied to maternal hemorrhagic shock remains unknown and provided the impetus for our study.MODS is the most common complication of shock and accounts for large parts of mortality. Therefore, the key point to treat patients with shock is to prevent MODS. It is now generally agreed that intestines are extremely vulnerable to the damages caused by bacterial translocation and ischemia during shock, which makes it the trigger organ of MODS. In addition, recent researches imply that fluid infusion has significant impact on the recovery of intestinal functions during resuscitation of shock. Improper fluid infusion leads to increase of mortality and morbidity.Data derived from our previous animal studies conferred the positive effects of hypotensive resuscitation on hemodynamics, decrease of hemorrhage and increased survival rate. But the precise mechanism of these benefits remains unknown. In order to explore the underlying mechanism, we propose to explore the protective effects of hypotensive fluid resuscitation for intestinal mucous barrier using pregnant rabbit model with uncontrolled hemorrhagic shock.ChapterⅠProtective effect of hypotensive fluid resuscitation on microcirculation in intestinal mucus in pregnant rabbit model with uncontrolled hemorrhagic shockOBJECTIVEData suggested that damage of intestinal mucus originally results from ischemia of intestinal villus and accumulation of oxygen free radical. The blood flow in intestinal mucus is a direct index of intestinal microcirculation, while serum concentrations of MAD and SOD represent the levels of lipid peroxidation and oxygen free radical respectively. Therefore, we demonstrate the protective effects of hypotensive fluid resuscitation on microcirculation in intestinal mucus in pregnant rabbit model with uncontrolled hemorrhagic shock by measuring the serum levels of MAD and SOD, as well as blood flow in microcirculation in intestinal mucus.METHODS1. Establishment of pregnant rabbit model with uncontrolled hemorrhagic shock 40 anesthetized New Zealand white rabbits at 15～25 days'gestational age were used for the establishment of animal model. The left common carotid artery was cannulated for continuous blood pressure monitoring and blood withdrawal, while the left jugular vein was cannulated for fluid administration. Then a lower abdominal midline incision was made and uterus was exposed. Baseline data were recorded when the vital signs stabilized, and animals began hemorrhaged at time point zero (TO). The experiment consisted of three phases:①shock phase (0～30min), exsanguinate animals via carotid artery to mean arterial pressure (MAP) of 40～45mmHg at an average rate of 2ml.kg-1.min-1 within 15min, all the blood were aspirated and heparinized for reinfusion;②simulated prehospital resuscitation phase (30-90min), one of the major artery in a gestational sac was transected, and then resuscitated according to the settled protocol. Free intra-abdominal blood was collected on preweighed pieces of cotton;③simulated hospital resuscitation phase (90～180min), At T90min, the laparotomy incision was reopened. The cannulas were then removed from the animals after ligation of the vessels, the incisions were closed. Venous infusions continued to maintaining an MAP of 80mmHg until the end of the experiment at T180min.2. Experiment protocol Animals were randomly divided into four groups (n=10 per group):①GroupⅠ(sham shock group, SSG), animals underwent anesthesia, cannulation and abdominal incision, without hemorrhage nor fluid resuscitation.②GroupⅡ(simple hemorrhage group, SHG), animals underwent hemorrhagic shock, but no resuscitation.③GroupⅢ(aggressive fluid resuscitation group, AFR), animals were aggressively resuscitated with Ringer's solution at a speed of 12ml/min, maintaining MAP as 80 mmHg from prehospital phase to the end of the experiment.④Group IV (low volume resuscitation group, LVR), hypotensive resuscitation at prehospital phase with Ringer's solution (4ml/min) to MAP of 60 mmHg followed by a raise of MAP to 80mmHg at hospital phase.3. Experiment parameters Blood flow of microcirculation in intestinal mucus (PERI ED Peri Flux 4001, Sweden) and vital signs (PcLab Bioinformation Processor) were recorded at T0, T30, T60 and T90 respectively. Blood samples were collected at the corresponding time points. Serum concentrations of MDA and SOD were measured by TBA method (kits purchased from ADE Ltd. USA.).RESULTS1. The hemodynamic parameters in LVR group were more stable throughout the experiment comparing with AFR group, indicating hypotensive fluid resuscitation asserts less influence on hemodynamics of pregnant rabbit with hemorrhagic shock. The vital signs in LVR group and AFR group at the end of the experiment were significantly different (respiratory rate:64 vs.72bpm; heart rate:232 vs.263bpm; MAP:82 vs.74mmHg; P<0.01).2. Variations of blood flow in microcirculations of intestinal mucus There was no significant difference concerning blood flow in microcirculations of intestinal mucus between groups at T0. Blood flows were significantly decreased at T30min (P< 0.01). Blood flow was undetectable in SHG due to continuous hemorrhage, while blood flows increased in AFR and LVR groups. Compared with AFR group, blood flow increased more significantly in LVR group. At T60min, the recorded values were 150.50 IPU in AFR group and 174.10 IPU in LVR group respectively (P< 0.01). At T90min, blood flow in LVR group increased to normal value (228.60IPU) while that in AFR group remained low (189.90 IPU) (P<0.01).3. Serum concentrations of MDA The normal range of MDA concentration is 6.0～9.0 mmol/L. There was no obvious change as to serum concentration of MDA in SSG, while MDA concentration in SHG continuously increased throughout the experiment. At T30min serum concentrations of MDA in each group were higher than the baseline (P<0.01). At T60min serum concentrations of MDA in AFR and LVR groups decreased, and the concentration in LVR group was lower than that in AFR group (9.15 vs 12.23 mmol/L, P<0.01).At T90min, serum concentrations of MDA in LVR group approached normal value (8.43 mmol/L) while that in AFR group remained high (11.03 mmol/L) (P<0.01).4. Serum concentrations of SOD The reference value of SOD in normal pregnant rabbit is about 180～220 mmol/L. There was no obvious change as to serum concentration of SOD in SSG, while SOD concentration in SHG continuously decreased throughout the experiment. At T30min serum concentrations of SOD in each group were lower than the baseline (P<0.01). At T60min serum concentrations of SOD in AFR and LVR groups decreased, the concentration in LVR group was higher than that in AFR group (165.91 vs 146.56 mmol/L, P<0.01). At T90min, serum concentrations of SOD in LVR group and in AFR group were 144.83 mmol/L and 117.72 mmol/L respectively (P<0.01).CONCLUSIONHypotensive fluid resuscitation significantly improved microcirculation and decreased damages of intestinal mucus in pregnant rabbit model with uncontrolled hemorrhagic shock. In addition, it inhibits lipid peroxidation by increasing serum SOD and decreasing MDA, therefore protects intestinal mucus from damage of oxygen free radicals.ChapterⅡInhibitory effects of hypotensive fluid resuscitation for translocation of bacteria in intestinal mucus in pregnant rabbit model with uncontrolled hemorrhagic shockOBJECTIVEThere is now general agreement that intestine is the critical organ during hemorrhagic shock, since it's the most vulnerable organ to ischemia-reperfusion, and the subsequent translocation of bacteria in intestinal mucus is the initial step for MODS. Based on these theories, we propose to explore the inhibitory effects of hypotensive fluid resuscitation for translocation of bacteria in intestinal mucus in pregnant rabbit model with uncontrolled hemorrhagic shock by measuring serum concentrations of endotoxin, TNF-αand intestinal bacterial colonies.METHODS1. Experiment protocol was the same as chapterⅠ.2. Blood samples were taken at T240min or when animals died. Serum concentrations of endotoxin were measured using Limulus test. Bacterial concentrations were determined by blood culture.3. Liver specimens and lymph nodes in mesoileum were taken at T240min or when animals died. Specimens were cultured for the measurement of bacterial colonies. In the meantime, swabs in the peritoneal cavities cultures were performed to testify the aseptic technique.4. Blood samples were collected at T0, T30min, T90min and T240min. Serum concentrations of TNF-a was measured by double-antibody sandwich ELISA method.RESULTS1. Cultures of swabs in peritoneal cavity There was no significant difference as to the culture results, indicating our operations had no interference with bacterial translocation and serum endotoxin.2. Bacterial colonies in liver specimens and lymph nodes in mesoileum Hemorrhagic shock significantly increased bacterial colonies in liver specimens and lymph nodes in mesoileum. Among the four groups, bacterial colonies in SHG were significantly higher than those in AFR and LVR. Bacterial colonies in liver specimens in AFR and LVR were 2.53×105CFU/g and 1.51×105CFU/g respectively (P<0.01). Bacterial colonies in lymph nodes in mesoileum in AFR and LVR were 4.32×105CFU/g and 3.25×105CFU/g respectively (P<0.01).3. Serum concentrations of endotoxin Normally the serum concentration of endotoxin in rabbit is lower than 0.5EU/ml. In current experiment, serum concentration of endotoxin in SHG was 3.42EU/ml, which was significantly higher than those in LVR (0.64EU/ml) and AFR (2.41EU/ml). Compared with that in AFR, endotoxin concentration in LVR significantly lower (P<0.01).4. Blood culture All samples in SHG grew bacterial colonies, while there were 4 and 1 positive results in AFR and LVR respectively.5. Serum concentrations of TNF-a There was no obvious change as to serum concentration of TNF-a in SSG, while TNF-a concentration in SHG continuously increased throughout the experiment. At T90min serum concentrations of TNF-a in AFR and LVR were higher than the baseline, TNF-a concentration in AFR was higher than that in LVR, but the difference was no significant. At hospital phase, the concentrations of TNF-a continuously increase, and the speed in AFR was faster than that in LVR (P<0.01). At T240min serum concentrations of TNF-a in AFR and LVR groups decreased, the concentration in LVR group was higher than that in AFR group, but the difference was no significant.CONCLUSIONCompared with aggressive fluid infusion, hypotensive fluid resuscitation significantly decreased translocations of intestinal bacteria, mainly E. coli. It also decreased serum concentrations of TNF-a and improved cellular immunity, as a result bacterial growth was inhibited.SUMMARYTo explore the feasibility of hypotensive fluid resuscitation for obstetrical hemorrhagic shock, we investigated protective effects of hypotensive fluid resuscitation on microcirculation in intestinal mucus in pregnant rabbit model with uncontrolled hemorrhagic shock by measuring the serum concentrations of endotoxin, MDA, SOD, TNF-a, as well as intestinal bacterial colony and blood flow in microcirculations in intestinal mucus.1. Pregnant rabbit exhibited typical signs of hemorrhagic shock 30min after hemorrhage. At this time, blood flow in microcirculation of intestinal mucus decreased significantly, serum concentrations of MAD, endotoxin and bacterial colonies in liver and lymph nodes in mesoileum increased, while serum concentration of SOD decreased, which accounted for the impaired function of intestinal mucus barrier in pregnant rabbit.2. Hypotensive fluid resuscitation significantly decreased translocations of intestinal bacteria, mainly E. coli, therefore decreased the serum concentration of endotoxin. In addition, it also decreased serum concentration of TNF-a and improved cellular immunity, as a result bacterial growth was inhibited.3. Hypotensive fluid resuscitation significantly improved the microcirculation in intestinal mucus and inhibited lipid peroxidation. As the SOD increased and MDA decreased, oxygen free radicals significantly decreased.4. Hypotensive fluid resuscitation at prehospital phase provided protective effects on the intestinal barrier, leading to significant decrease of complications followed by hemorrhagic shock. Therefore we speculate that it could be an effective way to resuscitate obstetrical patients with hemorrhagic shock even if further research is needed when extrapolating results obtained by these models to clinical settings.