The Empirical Study Of Limited Fluid Resuscitation In Pregnant Rabbits With Hemorrhagic Shock

The initial treatment of hemorrhagic shock often requires fluid resuscitation to replace intravascular blood volume lost due to injury. Clinical practices have advocated early and aggressive fluid resuscitation to restore vascular volume, organ perfusion and tissue oxygen delivery. However, mounting evidence from studies in experimental animals and clinical observation in recent ten years have demonstrated that aggressive resuscitation strategies are associated with cardiac and pulmonary complications, coagulation disturbances, immunological dysfunction, which results in increased blood loss and higher mortality.Based on these considerations, the concept of limited volume resuscitation has been proposed as a compromise strategy. Limited volume resuscitation allows the restoration of blood circulation with minimal fluid requirements and a modest increase in blood pressure, thus reducing the risk of additional blood loss due to continued bleeding or rebleeding. This resuscitation regimen has already shown efficacy in clinical trial of trauma and hemorrhagic shock.Postpartum hemorrhage is the leading cause of maternal mortality in our country. Due to special features of maternal hemodynamics, such as increased blood volume and hypercoaguiabie state, whether limited volume resuscitation could be apply to maternal hemorrhagic shock remains unknown and provided the impetus for our study.So we establish the pregnant rabbit model of hemorrhagic shock to mimic clinical situation, and test the efficacy of limited volume resuscitation on hemodynamics, immune system, coagulation system in pregnant rabbit with hemorrhagic shock.CHAPTER 1 ESTABLISHMENT AND EVALUATION OF PREGNANT RABBIT MODEL OF HEMORRHAGIC SHOCKOBJECTIVECompared with non-pregnant woman, the physiology of pregnant woman has following features: increased plasma volume and hypercoagulable state. While the difference of hemodynamics between pregnant and non-pregnant women with hemorrhagic shock remains unknown. So we establish a uncontrolled hemorrhagic shock model in pregnant rabbit to study hemodynamic changes of hemorrhagic shock in pregnancy.METHODS1. Determination of blood volume: the blood volume of each animal was determined by method of T-1842. Whole-blood samples were collected for HGB, HCT, PT, APTT, Fbg measurement.2. Surgical procedure Thirty New Zealand pregnant rabbits at 15-25 days' gestational age were fasted overnight, anaesthetized with an intravenous injection of 20% urethane(5ml/kg). Then the animals were placed in a supine position on an operating table and the left neck was prepared, scrubbed, and draped in a sterile manner. 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. After cannulation, lower abdominal midline incision was made and uterus was exposed. Baseline data were recorded after 15 rain stabilization, and animals began hemorrhaged at time point zero(T0). The experiment consisted of three phases, shock phase (0-30min), prehospital phase (30-90min), and hospital phase (90-180min). In the shock phase, 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, a medium vessel in a gestational sac was transected, and enclosed with aseptic sponges (for blood loss calculation). 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.3. Experimental protocol Animals were randomly assigned to three groups (n=10 for each group). GroupⅠ(sham shock group) served as normal control, animals were underwent anesthesia, cannulation and abdominal incision, but no hemorrhage nor fluid resuscitation. GroupⅡ(simple shock group) served as shock control, animals were underwent hemorrhagic shock, but no resuscitation. GroupⅢ(shock plus resuscitation group), animals were underwent hemorrhagic shock and resuscitation.4.Hemodynamic measurement Monitoring hemodynamic variables and respiration rate at T0, T30, T90, T180 and euthanizing one animal in each group at T240.5. Histological analysis A pathologist assessd the tissue damage under light microscope with double blinded method.RESULTS1.Blood volume of New Zealand rabbits increases from 4.43-6.66% of body weight in non-pregnant state to 5.76-8.51% of body weight in pregnant state, which has similar changes to gravidas in increased blood volume. HGB, HCT in non-pregnant rabbits are 150-300g/1, 0.32-0.52, while corresponding values in pregnant rabbits are 99-111g/1, 0.31-0.35. PT, APTT, Fbg in non-pregnant rabbits are 10-15s, 30-45s, 4.4-4.9g/1, while those in pregnant rabbits are 9.5-10, 25-40, 4.4-4.9. Data shown that pregnant rabbit have similar changes to gravida in terms of hemodilution and hypercoagulopathy.2.The hemodynamic parameters in sham shock group remained stable throughout the experiment, which meant no significant influence of surgical procedure.3.As expected, mean arterial pressure (MAP) in group two remained hypotensive after hemorrhage. The degree of hypotension was similar between group two and three at T30 (P＞0.05). MAP at T90, T180 in group three was significantly higher than group two due to resuscitation(P＜0.05).4.Assessment of kidney damage: no obvious pathologic changes was seen in sham shock group. More inflammatory cells infiltration in the glomeruli in the shock plus resuscitation group compared with simple shock group, and nephric tubules necrosis was only seen in the former groupCONCLUSIONThrough transected a medium blood vessel of gestational sac and blood withdrawal via carotid artery, we established a uncontrolled hemorrhagic shock model of pregnant rabbit, which mimicked the clinical situation and laid a foundation for studying hemodynamic changes of maternal hemorrhagic shock.CHARPTER 2 THE EFFECT OF LIMITED VOLUME RESUSCITATION ON HEMODYNAMICS IN PREGNANT RABBITS WITH UNCONTROLLED HEMORRHAGIC SHOCK OBJECTIVEA large body literature have shown that limited fluid resuscitation in uncontrolled hemorrhagic shock allows the restoration of blood circulation while reducing the risk of excessive bleeding and other complications associated with aggressive fluid resuscitation. So we test the efficacy of two resuscitation regimens in pregnant rabbit model of uncontrolled hemorrhagic shock, to explore the effect on the continuing hemorrhage, subsequent volume required in resuscitation and survival rate.METHODS1. Experimental protocol: Thirty pregnant New Zealand rabbits at 15-25 days' gestational age were randomly assigned to six groups (n=5 for each group), GroupⅠ(sham shock group) served as normal control, animals were underwent anesthesia, cannulation and abdominal incision, but no hemorrhage nor fluid resuscitation. GroupⅡ(simple shock group) served as shock control, animals were underwent hemorrhagic shock, but no resuscitation. GroupⅢ(shock plus aggressive fluid resuscitation group), animals were underwent shock and aggressively resuscitated with intravenous injection of Ringer solution to MAP of 80mmHg in prehospital phase. GroupⅣ,Ⅴ,Ⅵ(shock plus limited volume resuscitation group), animals were underwent shock and limited volume resuscitation with intravenous injection of Ringer solution at a rate of 4ml/min to MAP of 50, 60, 70mmHg respectively. The third phase is hospital phase, GroupⅢ,Ⅳ,Ⅴ,Ⅵreceived hemorrhage control by surgical intervention and adequately resuscitated to a MAP of 80 mmHg.2.Hemodynamics measurement: Hemodynamic parameters at T0, T30, T90, T120, T180 were monitored, subsequent volume resuscitation and survival status of 3h, 24h, 48, 72h were recorded respectively.RESULTS1.The difference in MAP among all groups at T90 was obviously protocol driven without practical value.2.Animals in groupⅣ,Ⅴ,Ⅵresuscitated to MAP of 50, 60, 70 mmHg in the prehospital phase had a higher and more stable MAP after adequate resuscitation in. the hospital phase. In contrast, animals in groupⅢthat received a large volume of fluid infusion to keep MAP at 80mmHg in the prehospital phase showed a significantly decreased MAP during the hospital phase. MAP at T180 in the limited volume resuscitation group VS aggressive fluid resuscitation group is 80 VS 72 mmHg, p＜0.05.3. The hemorrhage volume at T30 were comparable among all shock groups without significant difference, after hospital resuscitation, blood loss was significantly greater in aggressive fluid resuscitation group compared with limited volume resuscitation group (P＜0.05).4. The infused volume in prehospital phase and hospital phase were all significantly greater in aggressive fluid resuscitation group than limited volume resuscitation group(P＜0.05).5.No animal died within 180 min, the mortality of 72h in aggressive fluid resuscitation group was significantly greater than limited volume resuscitation group.CONCLUSIONCompared with aggressive fluid resuscitation,, limited volume resuscitation improved hemodynamics changes, decrease continuing bleeding and total fluid requirements, resulting in higher survival rate.CHARPTER 3 THE EFFECT OF LIMITED VOLUME RESUSCITATION ON HEMATOLOGY SYSTEM IN PREGNANT RABBITS WITH HEMORRHAGIC SHOCK OBJECTIVEPregnancy is considered a state of hypercoagulopathy owing to the progressive increase in the levels of all plasma coagulation factors (exceptⅪandⅩⅢ) and a decrease in the fibrinolytic activity due to a reduction in the level of the circulating plasminogen activator, which results in increased morbidity of deep vein thrombosis, pulmonary embolism and disseminated intravenous coagulopathy(DIC). Whether limited volume resuscitation regimen exacerbate this hypercoagulable state in maternal hemorrhagic shock remains unknown, so we test the feasibility of limited volume resuscitation on pregnant animals in hemorrhagic shock by observing the changes of prothrombin time (PT), activated partial thromboplastin time (APTT), hematocrit (HCT), content of fibrinogen (Fbg) and hemoglobin (HGB).METHODS1. Experimental protocol: fifteen pregnant New Zealand rabbits at 15-25 days' gestational age were randomly assigned to three groups (n=5 for each group), GroupⅠ(sham shock group) served as normal control, animals underwent anesthesia, cannulation and abdominal incision, but no hemorrhage nor fluid resuscitation. GroupⅡ(shock plus aggressive fluid resuscitation group), animals underwent shock and aggressively resuscitated with intravenous injection of Ringer solution at a rate of 12ml/min (results of the second chapter) to MAP of 80mmHg in prehospital phase. GroupⅢ(shock plus limited volume resuscitation group), animals underwent shock and limited volume resuscitation with intravenous injection of Ringer solution at a rate of 4ml/min to MAP of 60mmHg. The third phase is hospital phase, GroupⅡandⅢreceived hemorrhage control by surgical intervention and adequately resuscitated to a MAP of 80 mmHg.2. Whole-blood samples were collected at T0, T30, T90, T180, T240 for HGB, HCT, PT, APTT, Fbg measurement. RESULTS1. After hemorrhage, HGB, HCT and fibrinogen in GroupⅡandⅢmarkedly decreased, while APTT and PT gradually prolonged.2. PT, APTT at T0, 90, T180, T240 were significantly longer in aggressive fluid resuscitation group than limited volume resuscitation group (P＜0.05). Fbg concentration at T0, 90, T180, T240 was significantly lower in aggressive fluid resuscitation group than limited volume resuscitation group (P＜0.05). Fbg in limited fluid group returned to normal at T240.3. HGB, HCT at T90, T180, T240 was significantly decreased in aggressive fluid resuscitation group than limited volume resuscitation group(P＜0.05). HGB, HCT in limited fluid group returned to normal at T240.CONCLUSIONCompared with aggressive fluid resuscitation, limited volume resuscitation prevents severe hemodilution, maintains relatively proper HGB content and HCT for systemic oxygen delivery. Also, limited volume resuscitation alleviates coagulation disturbance by attenuating the decrease of Fbg concentration and prolongation of PT, APTT.CHAPTER 4 THE EFFECT OF LIMITED VOLUME RESUSCITATION ON SERUM CONCENTRATION OF TNF-a,IL-6 IN PREGNANT RABBITS WITH HEMORRHAGIC SHOCKOBJECTIVEHemorrhage and reperfusion induce systemic inflammatory response that leads to increased cytokine production, activation of the cellular immune response and subsequent damage of host organs by activated inflammatory cells. Previous studies have shown that limited volume resuscitation can inhibit this inflammatory response and reduce tissue damage in host organs. In the current study, we test if the beneficial effect of limited resuscitation can also be shown in pregnant rabbit model of hemorrhagic shock by examining the changes of serum concentration of tumor necrosis factor alpha(TNF-a), and interleukin(IL)-6.METHODS1. Experimental protocol: twenty pregnant New Zealand rabbits at 15-25 days' gestational age were randomly assigned to four groups (n=5 for each group), GroupⅠ(sham shock group) served as normal control, animals were underwent anesthesia, cannulation, abdominal incision, but no hemorrhage nor fluid resuscitation. GroupⅡ(simple shock group) served as shock control, animals underwent hemorrhagic shock, but no resuscitation. GroupⅢ(shock plus aggressive fluid resuscitation group), animals were underwent shock and aggressively resuscitated with intravenous injection of Ringer solution at a rate of 12ml/min (results of the second chapter) to MAP of 80mmHg in prehospital phase. GroupⅣ(shock plus limited volume resuscitation group), animals were underwent shock and limited volume resuscitation with intravenous injection of Ringer solution at a rate of 4ml/min to MAP of 60mmHg. The third phase is hospital phase, GroupⅢandⅣreceived hemorrhage control by surgical intervention and adequately resuscitated to a MAP of 80 mmHg.2. Heparined whole-blood samples were collected at T0, T30, T90, T180, T240, T480 for TNF-a, IL-6 measurement.3. Serum concentration of TNF-a, IL-6 were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer.RESULTSAfter hemorrhage, there was a trend to increased TNF-a, IL-6 levels in all groups except the sham shock group. Compared with aggressive fluid resuscitation, limited volume resuscitation attenuated the increase of serum concentration of TNF-a, IL-6 at T90, T180, T240, T480 (P＜0.05).At T480, TNF-a, IL-6 in the limited resuscitation group returned to normal, while those in aggressive fluid resuscitation were significantly higher than normal.CONCLUSIONHemorrhagic shock causes a striking increase in inflammatory cytokines. Compared with aggressive fluid resuscitation, limited volume resuscitation prevent inflammation by attenuating the increase of inflammatory cytokines, such as TNF-a and IL-6.SUMMARY1. Pregnant rabbit has similar physiology changes to gravida as increased blood volume, hemodilution and hypercoagulopathy, which suggests that the pregnant rabbit model of uncontrolled hemorrhagic shock can mimic the pathophysiology changes of maternal hemorrhagic shock.2. We successfully established clinically relevant model which mimic postpartum hemorrhagic shock and compared the efficacy of two resuscitations strategies. The major finding of our study is limited volume resuscitation enhance 20% of survival rate when compared with aggressive fluid resuscitation. The animals were resuscitated to to 50 or 60mmHg decreased blood loss and stabilized hemodynamics changes than those whose MAP maintained at 70mmHg in prehospital phase, but no difference in survival rate. For this reason, it is reasonable to conclude that hypotensive resuscitation to MAP of 50-60mmHg maybe an optimal critical pressure. Due to small sample size, the effects on major organ perfusion and microvascular changes remain unknown and need to be further studied.3. Elucidate the mechanism of superiority of limited volume resuscitation over aggressive fluid resuscitation in uncontrolled hemorrhagic shock.①modest increase in blood pressure in prehospital phase reduces blood loss and total fluid requirements, resulting in higher survival rate;②prevents severe hemodilution, maintains proper HGB and HCT, enhances systemic oxygen delivery, stabilize blood pressure, heart rate, respiration rate, which benefits for further resuscitation.③alleviates coagulation disturbance without exacerbate hypercoagulable state as indicated by Fbg, PT, APTT returned to normal at T240.④inhibits inflammatory response by attenuates an increase of inflammatory cytokines TNF-a, IL-6, prevents a decrease in lung wet/dry ratio and pulmonary edema, which suggests limited volume resuscitation alleviates the reperfusion injury induced by aggressive fluid resuscitation.In summary, we elucidate the efficacy and mechanism of limited volume resuscitation in pregnant rabbit of uncontrolled hemorrhagic shock.