The Effect Of Combination Therapy With Beta-blocker Plus Fluid Resuscitation On Survival And Myocardial Injury Of Restraint Stress And Crush Injury Model In Rats

Objective:Crush injury refers to the direct damage resulting from the crushing force,which mainly occurs on the limbs with skeletal muscle.Crush syndrome,also known as traumatic rhabdomyolysis refers to the systemic manifestation of breakdown of muscle cells with release of contents into the circulation after prolonged continuous pressure.Most of recent studies focused on pathophysiology and treatment of kidney damage after decompression.There is also research that suggests cardiomyocyte injury is caused by muscle crush injury at the early stage of decompression,but the mechanism is still unclear.Despite simply crush on limbs,CI-CS is a result of multiple factors such as hunger,immobilization stress,etc.If these factors have effects on myocardial injury are lacking of the scientific demonstration.Acute stress can activate adrenergic(or sympathetic)nervous system(ANS)and lead excessive release of catecholamine,resulting in overstimulation of beta adrenergic receptor(βAR).Sustained stimulation of β-AR has been shown to correlate with left ventricular dysfunction and heart failure.βAR blockades are competitive antagonists that block the receptor sites for the endogenous catecholamine epinephrine(E)and norepinephrine(NE)on adrenergic beta receptors of the ANS.In addition to lower blood pressure,they have a reductive effect on heart rate,myocardial contractility,myocardial oxygen consumption.They can also prevent the direct toxic effect of catecholamine on cardiac myocyte,improve LV function and reverse LV remodeling.The present study was to establish a rat model of combined restraint stress and crush injury to evaluate the indicative value of ECG on prognosis by monitoring changes of electrocardiogram during compression and observing living conditions.Meanwhile,serum and myocardial tissue levels of catecholamine,expression of myocardial β1AR were evaluated to prove that stress was related to myocardial injury in combined restraint stress and crush injury.Furthermore,we gave pretreatment to rats with βAR blocker(Bisoprolol)while conventional fluid resuscitation.Short-term and long-term indicators were both analyzed to evaluate its impact on survival rate and damaged cardiac myocyte.Part 1 The study of myocardial injury in rats with combined restrain stress and crush injuryObjective:To establish rat model of combined restraint stress and crush injury,and to prove the effect stress has on cardiomyocyte injury during compression by monitoring changes of electrocardiogram and observing living conditions.Methods: Fifty male Sprague-Dawley(SD)rats were randomly divided into two groups(n=25 per group): combined restraint stress and crush injury(model group),hunger and thirst(control group).ECG was performed for 72 h and survival rate was recorded till 7 days after decompression.Thirty-six male SD rats were randomly separated into six groups(n=6 per group): crush for 0h,6h,12 h,24h,48 h,72h respectively,blood and heart tissues were collected after decompression.Biochemical indexes and myocardial enzymes were measured by autobiochemistry analyzer(Hitachi 7180,Tokyo,Japan).Histologic changes in heart tissues were evaluated by Hematoxylin and Eosin staining(HE).Expression of β1AR in heart tissues was tested by immunohistochemical(IHC)staining.Serum and myocardial tissue levels of catecholamine were evaluated by high-performance liquid chromatography coupled with electrochemical detection(HPLC).Results:1 The changes of ECG: reduced heart rate,elevated ST segment,and tall-peaked T wave were observed in model group.No ECG changes were observed in control group except reduced heart rate 24 hours after compression.2 Survival rate: the whole survival rate of model group before decompression was only 55% and reached 25% 7 days after decompression.The survival rate of control group rats was 100%.3 Correlation analysis of ECG and survival: there was a positive correlation between survival time and the time ECG begins to change.4 The changes of biochemical indexes and myocardial enzymes: compared with 0h group,the serum BUN levels were significantly higher in 72 h group(P <0.01),the serum K+ levels increased from 48 h group(P <0.01),the serum LDH,CK,CKMB levels increased from 6h group(P <0.01),the serum AST levels increased from 12 h group(P <0.05)and ALT increased from 48 h group(P <0.01).5 HE staining results: the pathologic changes of heart tissues aggravated gradually with crush time extension in different time groups.6 IHC analysis of β1AR: the mean optical density(OD)value for β1AR decreased significantly in 6h group(vs.0h,P <0.01),then increased in 12 h group(vs.6h,P <0.01)and reached the first peak in 24 h group(vs.0h,P <0.05,vs.12 h,P <0.01),finally got to the peak in 72 h group(vs.0h,P <0.01,vs.48 h,P <0.01).7 Catecholamine changes in serum and heart tissues: the serum NE concentrations elevated from 12 h group(vs.0h,P <0.05),the serum E and DA concentrations elevated from 6h group(vs.0h,P <0.05).The whole concentrations of three catecholamine in heart tissues were lower,the trend of NE concentrations was similar to that in serum and no significant changes were observed in E and DA concentrations.Conclusions:1 Cardiomyocyte injury exists in the early stage of combined restraint stress and crush injury.2 Stress has important impact on cardiomyocyte injury caused by combined restraint stress and crush injury.Part 2 The effect of combination therapy with beta-blocker plus fluid resuscitation on survival and myocardial injury of restraint stress and crush injury model in ratsObjective:To evaluate the effect of β-AR blocker preconditioning(β-R)and conventional fluid resuscitation with 0.9% normal saline(SAL)on survival rate and cardiomyocyte injury of combined restraint stress and crush injury rats.Methods: One hundred seventy-five SD male rats were randomly separated into five groups(n=35 per group): Sham group(Sham),model group(crush injury),preconditioning group(β-R),conventional fluid resuscitation group(SAL),combination treatment group(β-R&SAL).20 rats were chosen for survival observation for 7 days after decompression and echocardiography at 1 day,7 days,30 days after decompression.15 rats were chosen for serologic examination and morphologic evaluation.Blood and heart tissues were collected before compression,1day and 30 days after decompression.Biochemical indexes and myocardial enzymes were measured the same way as in part 1.Histologic changes in heart tissues were evaluated the same way as in part 1.The fibrosis degree in heart tissues was evaluated with Masson’s trichrome staining and CVF was calculated.Results:1 Survival rate: survival in Crush injury group was 15%(3 of 20 rats).Survival in β-R group was 30%(6 of 20 rats).Survival in SAL group was 40%(8 of 20 rats).Survival in β-R&SAL group was 55%(11 of 20 rats).A log-rank test showed significant differences between β-R&SAL group and Crush injury group.2 The changes of biochemical indexes and myocardial enzymes: 1 day after decompression,compared with sham group,the serum AST,ALT,LDH,BUN,CR,CK,CKMB levels were significantly higher in model group,the serum AST,ALT,LDH,CK,CKMB levels decreased in β-R group.30 days after decompression,despite the serum LDH,CK,CKMB levels were slightly higher in model group,all indexes fell back to normal levels.3 HE staining results: 1 day after decompression,Sham group cardiomyocytes had clear structure,arranged neatly,stained evenly;Crush injury group showed cardiomyocytes local necrosis and a large number of inflammatory cell infiltration;disordered arrangement and twisted cardiomyocytes and part of myocardial fiber fracture were seen in β-R group;SAL group showed cardiomyocytes with disordered arrangement and abundant eosinophilic;β-R&SAL group showed cardiomyocytes with uneven staining but relative neat arrangement.30 days after decompression,pathologic damage in Crush injury group was still the most severe while different degree improvements were seen in other three treatment group,among which β-R&SAL group sustained the lightest damage.4 Masson’s trichrome staining results: 1 day after decompression,compared with sham group,CVF% was significantly higher in Crush injury group(21.43±3.82,P <0.01),compared with Crush injury group,CVF% decreased significantly in β-R&SAL group(17.52±3.74,P <0.01).30 days after decompression,compared with sham group,CVF% was significantly higher in Crush injury group(35.78±5.60,P <0.01),compared with Crush injury group,CVF% decreased significantly in all three treatment groups(β-R group,6.92±1.99,P <0.01,SAL group,9.59±1.86,P <0.01,β-R&SAL group,2.48±1.25,P <0.01).5 The results of echocardiography: M-mode echocardiogram of LV showed that ventricular wall motion weakened significantly 1 day after decompression and ventricular cavity increased apparently 30 days after decompression,which were improved 30 days after decompression in all three treatment group with varying degree.Compared with Sham group,LVM,LVVS and LVIDs of model group increased obviously from 7 days after decompression which suggested LV remodeling.30 days after decompression compared with model group,LVM,LVVs and LVIDs decreased significantly in β-R&SAL group,but no improvements were observed with FS%,EF% and LVPM.Conclusions:1 Survival rate can be improved by combination of β-AR blocker preconditioning and conventional fluid resuscitation in rats with combined restraint stress and crush injury.2 Combination of β-AR blocker preconditioning and conventional fluid resuscitation can alleviate myocardial fibrosis and improve impact on ventricular remodeling in rats with combined restraint stress and crush injury.