ECMO Resuscitating Prolonged Hemorrhagic Shock Alleviates Intestinal Mucosal Barrier Injury In Rabbits

Backgroud：The prolonged hemorrhagic shock could lead to the serious systemic inflammation,multiple organ failure (MOF) with a high mortality after resuscitation. The intestinalmucosal barrier damage,induced by the prolonged hemorrhagic shock, is considered to bethe ‘‘motor factor’’ driving MOF，and aslo causes a poor prognosis in critically illpatients.Therefore early and accurate assessment of mucosal barrier injury is clinicallyrelevant. Unfortunately, there is no serum marker that can accurately assess intestinalmucosal barrier injury induced by the prolonged hemorrhagic shock.In addition, the conventional fluid resuscitation still cannot effectively resuscitateprolonged hemorrhagic shock and then reduce intestinal musical barrier injury.Extracorporeal membrane oxygenation (ECMO) is a life-support technology.Over the pastfour decades，it has become a safe therapy for neonatal severe heart and lung failure,refractory cardiac arrest, early graft cardiac function failure, etc. Now, further experimentalstudies are needed to prove if ECMO improves the resuscitation of prolonged hemorrhagicshock and alleviates the intestinal mucosal damage.Objective:To construct a prolonged hemorrhagic shock rabbit model,the aim of this study was toexplore the impact of two degrees of hemorrhagic shock on intestinal mucosal barrierdamage, and to assess if serum DAO levels can reflect intestinal mucosal barrier injurysubsequent to the prolonged hemorrhagic shock. And we were further to assess if ECMOcan alleviate intestinal mucosal barrier injury by comparing ECMO to conventional fluidresuscitation of the prolonged hemorrhagic shock rabbit. Methods:1. The construction of prolonged hemorrhagic shock rabbit model and theevaluation of the severity of intestinal mucosal injuryThe aim of the first part of the study was to assess if serum DAO levels can reflectintestinal mucosal injury subsequent to prolonged hemorrhagic shock. Thirty New Zealandwhite rabbits were randomly divided into three groups: a control group, a medium bloodpressure (BP) group (exsanguinated to a shock BP of50to41mm Hg), and a low BP group(exsanguinated to a shock blood pressure of40to31mm Hg), in which the shock BP wassustained for180min prior to fluid resuscitation. The blood pressure changes wererecorded with an invasive arterial blood pressure monitoring device, andthe arterial lactatelevelswere recorded with portable blood gas analyzer.The expression levels of serumTNF-α and intestinal tissue myeloperoxidase (MPO) were analyzed by ELISA, andintestinal mucosal injury Chiu’s score were evaluated by pathological HE staining.Theexpression levels of Claudin-1and intercellular adhesion molecules (ICAM-1) in intestinaltissue were respectively detected by immunofluorescence staining, and theirfluorescenceintensities were estimated by Image-Pro Plus7.0software.2．ECMO resuscitating prolonged hemorrhagic shock alleviates intestinal mucosalbarrier injury in rabbitsIn the second part of the study, in order to investigate the effect of ECMO resuscitationon alleviating intestinal ischemia–reperfusion injury in a prolonged hemorrhagic shockrabbit model，thirty New Zealand white rabbits were randomly divided into three groups:control group (Control), conventional fluid resuscitation group (Group1), andECMOresuscitation group (Group2). The prolonged hemorrhagic shock model wasestablished keeping the arterial blood pressure from40to31mmHg for3h through thefemoral artery bleeding, and performing the resuscitation for2h by conventional fluidresuscitation and ECMO resuscitation respectively. Chiu’s score of intestinal injury, serumlactate and TNF-α levels and intestinal mucosa myeloperoxidase (MPO) activity,intercellular adhesion molecule (ICAM-1) and Claudin-1expression were detected.Results:1.The prolonged hemorrhagic shock animal model was constructed with New Zealand rabbits, in which the hemorrhagic shockmaintenance phasewassuccessfully prolonged to3hours and the shock BP was maintained above30mmHg. Before the shock, MAP amongthe control group, the medium BP group and the low BP group were not significantlydifferent. MAP in the control group was relatively stable, fluctuating from108mmHg to96mmHg. The mean arterial pressure (MAP) decreased to the target shock BP of50to41mmHg in the medium BP group and40to31mmHg in the low BP group. MAP during theshock maintenance phase was42.8±4.7mmHg in the medium BP group and35.8±5.6mmHg in the low BP group. After resuscitation, MAP in the medium BP group wassignificantly lower than that in the control group（70.0±4.8mmHg vs.101.1±3.3mmHg，P<0.01）. And MAP in the low BP group was also significantly lower than that in thecontrol group (41.7±5.1mmHg vs.101.1±3.3mmHg，P<0.01). MAP in the low BP groupwas also significantly lower thanthat in the medium BP group (41.7±5.1mmHg vs.70.0±4.8mmHg，P<0.01). The recovered MAP after resuscitation in the low BP groupwasworse than that in the medium BP group.2.The severity of prolonged hemorrhagic shock rabbits. No significant differencesinserum TNF-α and arterial blood lactates were observed between in the medium BP groupand in the low BP group before the shock. Compared withthe medium BP groupbefore andafter resuscitation, serumTNF-αlevels in the low BP group were significantlyincreasedrespectively (0.319±0.016ng/ml vs.0.215±0.009ng/ml, P<0.01；0.626±0.0429ng/ml vs.0.362±0.020ng/ml, P<0.01). Compared with the medium BP group before andafter resuscitation,arterial blood lactate levels in low BP group were significantly higherrespectively (18.97±1.52mmol/L vs.14.25±0.80mmol/L, P<0.01；13.58±1.27mmol/L vs.10.29±1.02mmol/L, P<0.01).3. The degrees of intestinal mucosal barrier damage in the prolonged hemorrhagicshock rabbits.Comparedwith themedium BP group, intestinal mucosal damage Chiu’sscoresin the low BP group were significantly higher (3.5±0.53vs.2.3±0.67, p<0.05), andimmunofluorescence intensity of Claudin-1in the low BP group was significantly lower(87.39±8.78vs.142.61±10.43, p<0.05), and immunofluorescence intensity of ICAM-1inthe low BP group was significantly increased (297.17±6.15vs.191.34±11.02, p <0.05), and MPO levels in the low BP group were significantly increased (0.911±0.068IU/g vs.0.615±0.047IU/g, P<0.01).4. The levels of serum DAO in the prolonged hemorrhagic shock rabbits. Beforeresuscitation, the serum levels of DAO in the medium BP group and the low BP group werehigher than thatin the control group respectively (0.153±0.020IU/L and0.252±0.026IU/Lvs.0.107±0.008IU/L, P<0.01). After resuscitation, the levels of serum DAO in the mediumBP group and the low BP group were also higher than that in the control group (0.337±0.037IU/L,0.670±0.085IU/L vs.0.110±0.009IU/L, P <0.01).Further, before andafter resuscitation, the serum DAO level in medium BP group moderate were lower thanthat in low BP group respectively (0.153±0.020IU/L,0.252±0.026IU/L, P<0.01;0.337±0.037IU/L vs.0.670±0.085IU/L, P<0.01). Statistical analysis showed that afterresuscitation, the levels of serum DAO had a positive correlation with serum TNF-α(R=0.970, P <0.01), and also with Chiu’s score(R=0.601, P <0.01).5.The effect of ECMO resuscitating the prolonged hemorrhagic shock rabbits.Nosignificant differences in MAP, serum TNF-α and arterial blood lactate were observedbetween in the ECMO group and in the conventional fluid resuscitation group before theshock. Compared with the conventional fluid resuscitation group, MAP in the ECMOgroupwere significantly increased after resuscitation (63.3±5.6mmHg vs.42.0±5.1mmHg,P<0.05), butarterial blood lactate levels in the ECMO group were significantly lower(10.53±0.85mmol/L vs.13.63±1.22mmol/L, P<0.05), and serum TNF-α levels in theECMO group were also significantly lower (0.584±0.045ng/ml vs.0.622±0.047ng/ml,P<0.05).6. The degrees of the mucosal injury after ECMO resuscitating the prongedhemorrhagic shock.Compared withconventional fluid resuscitation group, the Chiu’s scorein the ECMO group were significantly decreased (2.0±0.67vs.3.6±0.52, p<0.01), andintestinal tissue MPO levelsin the ECMO group were significantly decreased (0.685±0.067IU/g vs.0.922±0.064IU/g, P<0.01), and immunofluorescence intensity of ICAM-1in theECMO group wasalso significantly lower (160.04±9.56vs.236.72±14.12, P<0.05), butimmunofluorescence intensity of Claudin-1was significantly increased (149.07±8.30vs. 97.62±9.80, P<0.05).Conclusion:1. The prolonged hemorrhagic shock rabbit model was successfully constructed.Thehemorrhagic shockmaintenance phase was prolonged to3hours before resuscitation. Themodel was created by bleeding through the artery on the base of the fixed-pressure system.The model building method is reliable and reproducible. Our experiencesare that during theshock maintenance phase, MAP should be maintained at30mmHg above.2. Severe systemic inflammation and mucosal barrier system damage occurred inprolonged hemorrhagic shock rabbits. In the medium BP group and thelow BP group,serum TNF-α levels and arterial blood lactate levels were significantly increased, andintestinal structural damage was aggravated, and Claudin-1expression levels weresignificantly reduced, and intestinal tissue MPO, ICMA-1expression levels weresignificantly increased. Meanwhile, in two different BP levels (50-41mmHg and40-31mmHg), the lower shock BP was maintained, the more serious leukocyte infiltration oftissues andsystemic inflammationoccurred, andeventually the more serious mucosal barrierdamage occurred.3. Serum DAO as a serum marker can reflect the degrees of intestinal mucosal barrierdamage andprolonged hemorrhagic shock severity at the same time. In the prolongedhemorrhagic shock rabbit model, both serum DAO levels in the medium BP group and inthe low BP group were significantly higher than that in the control group.Serum DAOlevelin low BP group was significantly higher than that in the medium BP group.Statisticalanalysis of the experimental data showed that serum DAO had a positive correlation withChiu’s score and TNF-α respectively.4. The prolonged hemorrhagic shock rabbits in the low BP group were resuscitated byECMO and conventional fluid.ECMO resuscitation had better results than conventionalfluid resuscitation. MAP in ECMO group recovered to a higher level,and serum lactatelevels were significantly reduced, and serum TNF-α levels were also significantly reduced.ECMO resuscitation improved tissue perfusion and reduced systemic inflammation. 5. After the prolonged hemorrhagic shock rabbits in the low BP group wereresuscitated by ECMO and conventional fluid respectively, ECMO alleviated themucosalbarrier injury.After ECMO resuscitation, Claudin-1expression in intestinal tissue wassignificantly increased,the levels of ICAM-1and MPO expression were significantlyreduced. Therefore, ECMO mitigates systemic inflammatory response and reduces themucosal barrier injury at the same time.6. The possible mechanism of ECMO alleviating intestinal mucosal barrier injuryafter ECMO resuscitation may be related to ECMO reducing the systemic inflammation andleukocyte infiltration of intestinal tissue.