Effects Of Pure Oxygen And Room Air Resuscitation On Myocardial Cells Of Asphyxial Neonatal Rats

Objective:To establish the animal model of neonatal rats with asphyxial myocardial damage,and compare the effects of pure oxygen and room air resuscitation on the damage of myocardial cells of asphyxial neonatal rats by detecting the related indexes and ultrastructual changes of myocardial cells.The results may clinically used as a scientific theoretical basis for early resuscitation program against neonatal asphyxia.Method:7-day-old,cleaning SD(Sprague Dawley) rats 30,weight 12.5±1.0g.(from Guangzhou Baiyun Shima Experimental Animal Center).Purchased on the 5th day after birth,adapting to the environment for 2 days.Neonatal rats were weighed before modeling and given numerical olders according to their weight.30 neonatal rats were randomly divided into 24h group(resuscitation after 24h,n=15) and 72h group(resuscitation after 72h,n=15).Each group was divided into three sub-groups:normal control group(n=5);atmospheric pressure oxygen resuscitation group(normal pressure 100%O2,or pure oxygen Group,PO,n=5);air resuscitation group(21%O2,or air group,RA,n=5).Before the experiments a low oxygen chamber was placed into water bath at 36℃～37℃,and the cabin temperature was controlled around 36℃.PO Group and the RA group of neonatal rats were placed into the preheated chamber and ventilated the chamber with mixed gas of 8%O2 and 92%N2 at a flow rate of 1.5L/min,continuing 2.5h.Then,put the rats of PO group into the PO chamber immediately,inflowed with atmospheric 100%O2,at the velocity of 5L/min.The environment of oxygen chamber was the same with the modeling chamber.The rats of RA group were placed into RA chamber in the same room.Each group was resuscitated for 30min, respectively.Thereafter,put all neonatal rats back to the mother rats,decapitated executed the neonatal rats at the time point of 24h,72h after resuscitation,and took the heart conventionally.Myocardial tissue was embedded in epoxy resin and paraffin conventionally and made into paraffin sections and electron microscopy specimens.Histopathological examination was done through HE staining,apoptotic cells were continued through TUNEL method and apoptosis index was calculated.Expression intensity of Bcl-2 and Bax protein was detected by immunohistochemistry and image analysis system.The ultrastructural changes of myocardial cells were observed transmission electron microscopically.Results:1.Behavior Observation:Behavior changes during the phase of hypoxia model:neonatal rats showed increasingly cyanotic lips,gradually accelerated respiration.The gray and white skin became dark red. Convulsion occurred sometimes and it was difficult to complete righting reflex.Such acute hypoxia performance continued to 2.5h at the end of experiments.No neonatal rats died.Behavior during the resuscitation phase:In the PO resuscitation group,the neonatal rats showed systemic fibrillation and their skin became rosy gradually.In the RA resuscitation group,the neonatal rats gazed up,in a stiff trance,with gray and white skin,and the cyanosis disappeared slowly.2.pathological changes of myocardial tissue in HE staining:Normal control group:there were no obvious changes in myocardial cells,myofibrils were arranged in parallel with each other,and displayed branches.RA group:The myocardial cells showed patchy or focal necrosis.Reactive neutrophile granulocytes infiltrated between necrotic cells.PO group:damage of myocardial cells was less serious than that of RA group.Most myofibrils only showed swelling or fracture,Myocardial mesenchyme and small vein congested obviously.Mapholagically each group did not show significant differences at two different time pionts.3.Analysis of myocardial apoptosis index:Compared with normal control group,many TUNEL positive staining myocardial cells could be observed in PO and RA group,which showed smaller cell sizes and brown stained nuclei.In PO24h and 72h group,cell apoptosis index was significantly higher than the corresponding normal control group(P＜0.01).In RA24h and 72h group,cell apoptosis index was higher than the corresponding PO group(P＜0.01).The cell apoptosis indexes of 24h were higher than that of 72h group in either PO group or RA group(P＜0.05).4.BCL-2 and BAX protein expression in myocardial cells:There was a small amount of positive expression of Bcl-2 and Bax protein in the normal control group,which showed dark brown masses distributed asymmetrically in myocardial cytoplasma.Compared with normal control group,the expression intensity of Bcl-2 protein significantly increased in PO group(P＜0.01).The expression intensity of Bax protein in PO group was much higher than that of normal control group(P＜0.01).Compared with PO group, the expression intensity of Bax protein in RA group increased significantly(P＜0.01),but the expression intensity of Bcl-2 protein decreased significantly(P＜0.01).The time of each group did not have significant differences at two different time points.(P＞0.05).5.Ultrastructural changes in myocardial cells:Normal control group:Myocardial cells had membrane integrity,paralleled myofibrils, mitochondrial membrane was intact and the ridges ranged longitudinally or like concentric circles.Chromatin in myocardial nucleus distributed uniformly.PO 24h group:Myocardial cell membrane thickened and fuzzy,myofibrils disarranged.Some myofilament fractured,local small-scale dissolved lesions were visible.Mitochondrial were swollen.The cristal widened and some mitochondria becamed vacuolated.Nuclei were irregular,and some were jagged.PO 72h group:Myocardial cells still showed some injuries,but in a lesser extent,than 24h group. RA24h group:The extent of myocardial cell damage were significantly heavier than the PO group.Myocardial cells were seriously edematous,with unclear boundaries.Myolemma ruptured,defected.Nuclei were rounded,myofibrils fractured,dissolved,forming a large number of muscle dissolved lesions.Mitochondrial were diffusely swollen,with a ruptured outer membrane and cristal,and sometimes ruptured,dissolved and vacuolized.The nuclei were swollen,chromatin stained slightly.In RA72h group,changes in myocardial cells was slightly reduced than those of 24h group.Conclusions:Under this experimental condition,apoptosis of myocardial cells after RA resuscitation was more serious than that after PO resuscitation,especially after 24h.RA resuscitation promoted increased expression of Bax gene,and PO resuscitation could make increased expression of apoptosis suppressing gene of Bcl-2,suggesting that high concentrations of oxygen have some protective effect on asphyxial myocardial cells after resuscitation.Damage on the ultrastructure of myocardial cells were more serious after RA resuscitation than those after PO resuscitation.Typical pathological changes appeared 24h after resuscitation in both groups.A certain degree of regeneration might occurr 72h after resuscitation.