| Background:Neonatal hypoxic-ischaemic encephalopathy(HIE) is a severe complication of asphyxia that occurs before,during,or after birth which can result in death or neurological damage.The main lesions of brain tissue are edema,necrosis, and apoptosis.Because of complex pathogenesis of HIE,Supportive care targeting the symptoms can not improve children's brain damage and long-term prognosis fundamentally.Furthermore,the current neuro-protective therapy is still at the exploratory stage,searching for more effective treatment of HIE is still an important domestic and abroad issue.Hypoxic-ischemic brain injury is an evolving process,which begins after the attack and extends into the reperfusion interval after resuscitation,The pathogenesis of HIE includes circulating factors(cerebral blood flow disorder),metabolic factors (increased anaerobic metabolism,reduction of ATP),as well as chemical factors (increase of excitatory amino acids,intracellular calcium accumulation,and generation of oxygen free radicals and inflammatory factors),which is the foundation of the neuro-protective therapy for brain damage.The study found that Cyclic Adenosine-3'5'-monophosphata(cAMP) involved in a variety of nervous system diseases' mechanisms and also play an important role in damage and repairing of the nervous system.cAMP is a nucleotide derivatives,the "second messenger",it acts as a mediating factor by transferring hormones and transmitters in cell.Intracellular cAMP is synthesized by degradation of phosphodiesterase(PDF) while adenylate cyclase(AC) is the catalytic agent,so the stability of cAMP in cells depends on functional status of both agents.The changes of intracellular cAMP concentration affect a variety of intracellular signal transduction pathways to regulate protein activity,gene expression and cell function,also promote cell differentiation,growth,metabolism and the process of apoptosis.The study found that cAMP plays an important role in the nervous system damage:①Impact of nerve cell membrane protein structure and function;Involve in synaptic transmission;②Regulation of systolic and diastolic function in cerebrovascular;③Prevent calcium influx;④Inhibit variety of damaging intracellular cytokine's expression and production in Pathological state,Such as tumor necrosis factor,interleukin,starch-like substances;⑤Anti-apoptosis and promoting the recovery of neurological function;⑥Involved in learning and memory process.Animal experiments show that ischemia-reperfusion injury can lead to decreasing of the level of cAMP and AC mRNA in cerebral cortex and hippocampus;Spinal cord injury also caused the level of cAMP reduced in partial damage and the corresponding cerebral cortex.Clinical study found that CSF cAMP concentrations decreased in infants with asphyxia.Low CSF cAMP levels are associated with poor neurologic outcome,cAMP is one of the key factors in regulation of the central nervous system function,Its reduction may cause some dysfunction of the nervous system and trigger the occurrence of disease.It can be envisaged that after hypoxic-ischemic(HI) injury,exogenous cAMP is added or the drugs which can delay the degradation of cAMP are given in time,these methods could increase cAMP level to safe border and prevent imbalance of the cAMP signaling pathway from occurring at an early stage,then promote neuronal injury recovery.Studies show that giving exogenous db-cAMP and PDE IV inhibitor rolipram after spinal cord injury can promote axonal regeneration,the formation of myelin and nerve function.Another study shows that after cerebral ischemia,rolipram enhances hippocampal neurons regeneration,and promote the survival of newborn neurons.Meglumine cyclic adenylate(MCA) is a new type of cAMP derivatives,the ligand meglumine can increase the fat-soluble cAMP,making its entering the cells easier and play the role.MCA has been widely used in clinical practice for the treatment of heart failure and has certain therapeutic effect for myocardial injury after neonatal asphyxia. Some people observed that in adult rat after cerebral ischemia-reperfusion injury MCA pretreatment can reduce infarction and improve the prognosis.However, whether MCA has the protective effect on neonatal or newborn animals with hypoxic ischemic brain injury has not been reported domestically and abroad.Aminophylline is theophylline and ethylenediamine complex salt,non-selective PDF inhibitors and can increase intracellular cAMP levels.Aminophylline is widely used in clinic.Its pharmacological effects including:expansion of bronchoalveolar, cardiotonic,diuretic,anti-inflammatory,treatment of repeated apnea of prematurity and cerebral hemodynamics regulation,etc.Study shows that theophylline and aminophylline can improve nerve injury after stroke,reduce ischemic brain edema and brain damage;Research also shows that theophylline has a protective effect on neonatal rats with hypoxic-ischemic brain injury.Theoretically,MCA and aminophylline combination may has a synergistic effect in raising the level of intracellular cAMP,vasomotor regulation of brain function, anti-inflammatory effects and improving cardiac functionObjective:In this study,We use the model of neonatal rat hypoxic-ischemic brain damage(HIBD) trying to understand the changes of cAMP after HIBD and the relationship between the level of cAMP and brain injury;Preliminarily study the neuro-protective effect of exogenous cAMP drugs(MCA),aminophylline and two drugs in combination in neonatal rats after HIBD.Designed to provide new ideas and drugs for the clinical treatment of neonatal HIEMethods1.HIBD model and success criteriaRat pups were anaesthetized by ether and duration of anaesthesia was less than 5min.Hypoxic ischemia was induced according to the Levine-Rice model.A median incision was made in the neck.The left common carotid artery was dissected and ligated with a 4-zero silk suture.After the wound was sutured,the animals were allowed to have 1-2h recovery and feeding period.Except for the sham group,rats were then placed in a chamber and exposed to a continuous flow of 8%oxygen-92% nitrogen for 2h.The animals of the sham group received median neck incision. Neither ligation nor hypoxia was performed.The chambers were partially submerged in a water bath at 37℃to maintain a constant thermal environment.Rats of experimental group were assessed according to Zeal Longa standard after HI,without neurologic deficit was marked as 0 point;flexion of left anterior claws when hanging tail as 1 point;circling to left while walking as 2 points;difficulty in walking and falling down to left as 3 points;unconscious mind as 4 points.The rats assessed 1to 4 point were successful HIBD rats.2.Experimental grouping2.1 The changes of cAMP content in brain tissue36 7-day-old SD rats were randomly divided into sham operation group,2h group, 24h group,48h group,72h group and 168hgroup after HI,every group containing six rats;all the animals of experiment group were injected with saline intra-peritoneally after HI,4ml/Kg,2 times/day.2.2 The effect of MCA and Aminophylline on neonatal rats after HIBD180 7-day-old SD rats were randomly divided into sham operation group,HIBD group,MCA group,aminophylline group,MCA + aminophylline group(Combined Group);Each group contain 36 rats.After model successfully build,MCA group, aminophylline group and combined group were given drugs by intra-peritoneal injection,2 times/day,three days.MCA Group were given MCA 2mg/Kg, aminophylline group aminophylline 2mg/Kg,combined group Aminophylline 2mgl/Kg + MCA2mg/Kg,HIBD model group just the same amount of saline.3 Main observation target3.1 The changes of cAMP content in brain tissueAnimals in each group,Oh(sham group),2h,24h,48h,72h,168h after HI,were decapitated,10%tissue homogenate are made of brain,Measure the cAMP content of brain tissue in each group with double antibody sandwich ABC-ELISA method.3.2 The cAMP content in brain tissue after Drugs interfereAnimals in each group,24h,48h,72h after HI were decapitated,10%tissue homogenate made of brain.Measure the cAMP content of brain tissue in each group with double antibody sandwich ABC-ELISA method.3.3 brain tissue water content and the extent of brain damage;3.4 Measure brain oxidative metabolites MDA and antioxidant enzymes SOD content, 72h after HI,rats were decapitated and 10%tissue homogenate were made of brain, MDA was measured with modified thiobarbituric acid method;SOD with xanthine oxidase enzymatic determination method. 3.5 Neurological deficits score:Animals in each group were assessed according to Zeal Longa standard,2h,24h,72h after HI3.6 Observe histological changes in the cerebral cortex with hematoxylin - eosin(HE) staining method.Results1 The changes of cAMP content in brain tissue after HI:Multiple comparisons among groups showed that:brain cAMP content of sham group is the highest;Compared with the sham-operated group,the cAMP content in 2h group,24h group,48h group,and72h group and 168hgroup after HI were significantly decreased,and the difference had statistical significance(P=0.000); Compared with the 2h group,the cAMP content in 24h group,48h group,72h group and 168h group had rising trend,and the difference had statistical significance(P= 0.000).2 The cAMP content in brain tissue after Drugs interfere24h,the cAMP contents of HIBD group,aminophylline group,MCA group,and combined group were significantly different(F=960.203,P=0.000);cAMP content of aminophylline group,MCA group,and combined grouop were higher than HIBD group,cAMP content of combined group was higher than the aminophylline group, MCA group,cAMP content between aminophylline group and MCA group has no significant difference.48h,among the five groups the cAMP content were significantly different(F=749.353,P=0.000),cAMP content in MCA group and the combined group were higher than those of HIBD model group,cAMP content between aminophylline group and HIBD group has no significant difference,cAMP content of the combined group was higher than that of aminophylline group, aminophylline group was lower than MCA group;72h,among the five groups the cAMP content were significantly different(F=122.398,P=0.000),cAMP contents of HIBD group,aminophylline group,MCA group and combined Group were higher than that of HIBD group,but were lower than that of sham-operated group,cAMP content in combined group was higher than that of aminophylline group,MCA group, AMP content of aminophylline group was lower than that of MCA group.3 The determination of brain tissue water content and the extent of brain damage:Comparison of brain tissue water content:The result of multiple comparisons among groups showed that water content of aminophylline group was the lowest; compared with the sham-operated group,brain water content of aminophylline group, and combined group had no significant difference(P=0.908 and P=0.056),but brain water content of HIBD Group was significantly higher than that of MCA group(P=0.000 and P=0.003);Compared with HIBD model group,brain water content of MCA group,aminophylline group,and combined group significantly decreased,(P=0.000);Compared with the combined group,brain water content of MCA group was significant higher(P=0.013),brain water content of aminophylline group decreased(P=0.044).Comparison of the extent of brain injury:the result of multiple comparisons among groups showed that degree of brain damage of sham-operated group was the lightest; Compared with the sham-operated group,HIBD group,MCA group,aminophylline group,and combined group all had varying degrees of brain damage,and the extent of brain injury of combined group was the lightest,the data showed statistical significance(P=0.0000);and compared with HIBD group,extent of brain damage of the drug treatment group(MCA group,aminophylline group,combined group)decreased,the results had statistical significance(P=0.000);but brain injury between aminophylline group and MCA group has no significant difference(p=0.217). 4 MDA content and SOD activity of brain tissue in each group:MDA content in brain tissue:the result of multiple comparisons among groups showed that:MDA content of HIBD group was the highest;Compared with the sham-operated group,MDA content of combined group has no significant difference (P=0.385),MDA levels of HIBD group,MCA group,and aminophylline group were all significantly increased,and the results had statistically significance(P＜0.05);Compared with HIBD group,MDA content of MCA group,aminophylline group,and combined group significantly decreased,and the difference was significant (P=0.000);but the MDA content between aminophylline group and the MCA group had no significant difference(P=0.875).Brain tissue SOD content:The result of the multiple comparisons among groups showed that SOD content of HIBD model group was the lowest;Compared with the sham-operated group,SOD content of combined group had no significant difference (P=0.326),SOD content of HIBD group,MCA group,and aminophylline group were significantly lower,the difference was significant(P＜0.05);and compared with HIBD group,SOD content of MCA group and aminophylline group and combined group increased significantly,the difference was statistically significant(P=0.000). Compared with the combined group,SOD content of MCA group and aminophylline group significantly lower(P＜0.05).5 Effect of drugs on neural function in rats with hypoxic-ischemic brain injury:Neurological deficits scores of Sham-operated group at 2h,24h,72h all were 0 points(no neurological deficits),2h,the scores of HIBD Group,aminophylline group, MCA group,and combined group had no significant difference(F=0.524,P=0.475); 24h,the scores of HIBD group,aminophylline group,MCA group,and combined group were significantly different(F=14.832,P=0.001),the scores of aminophylline group,MCA group and the combined group were lower than those of HIBD group,the scores of combined group,aminophylline group and the MCA group had no significant difference;72h,the scores of HIBD group,aminophylline group, MCA group,and combined group were significantly different(F=24.653,P=0.000), and the scores of aminophylline group,MCA group and the combined group were lower than HIBD group,combined group's score was lower than the aminophylline group's and MCA group's.6 Histological results of Brain tissue:The right side of the brain of Sham-operated group and HIBD group:the number of nerve cells was large,the layer of cortical was clear,In addition,cell morphology and structure was clear,and neatly arranged;neuronal cytoplasm were rich.HIBD group:the structure of the brain cortex in the left side could be seen disordered,the organizational structure was loose,and there were many scattered cavities(necrosis liquefaction zone),in addition,neuronal nuclear pyknosis,cell shrinking deformation could be seen.Neurons with the normal patterns disappeared.Aminophylline group: the number of nerve cells decreased,structure of the cortex was clear in some degree, cell morphology became unclear and irregular,showing a number of neuronal nuclear pyknosis,cell shrinking deformation.MCA Group:the number of neurons was relatively large and cortical structure was relatively clear,cell morphology and structure was clear and neatly arranged.We can see a little of neuronal nuclear pyknosis,cell shrinking deformation.Combined group:the number of neurons was large,cortical structure was clear,and patterns of neurons was regular.But still we could see shrink in individual cells.Conclusions:1 The experiment confirmed that cAMP metabolic disorder happens after HI,two hours after HI,brain cAMP level decrease significantly(to about 25%of normal), then gradually increase,but still is lower at 168h(70%of normal);demonstrating that the body has certain ability of self-repairing after HI,but the effect is limited and took more time.2 Our study showed that MCA intervention can significantly increase the level of intracellular cAMP,reduce the brain edema and reduce the generation of oxygen free radicals,effectively improve neurological motor function,illustrate that CAMP metabolism disorder may be involved in the pathogenesis of hypoxic injury,3 Study found that as PDE inhibitor,aminophylline alone can not significantly increase the cAMP level of brain,but can help cAMP-type drugs to raise the level of cAMP;Aminophylline alone also has capacity to reduce the brain tissue damage, suggesting that aminophylline may has multiple pharmacological effects(such as anti- inflammatory,diuretic,regulating cerebral hemodynamics,improve breathing, etc.) therefore plays a role in brain protection4 Joint application of the two drugs,with the exception of brain water content indicators,other indicators are better than single-drug group,tips combined drug application in hypoxic-ischemic brain injury may has good synergic effect.
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