| Background and ObjectiveHypoxia ischemia refers to different degrees of the brain tissue oxygen deprivation and decreased blood flow, even interrupted,which caused by various risky factors in perinatal period,and can result in ischemic brain injury, it can contribute to neonatal death and cerebral palsy, epilepsy,mental retardation and other long-term sequelae,so early intervention for the treatment of HIBD had been the reasearch focus of many scholars.Caffeine belongs to the family of drugs known as the methylxanthines, and it is well known for treatment of apnea of prematurity, it can pass through the blood-brain barrier,and act as an antagonist of adenosine receptors exert many of its biological effects. Caffeine can not only prevent and treatm of dysplasia bronchopulmonary dysplasia,but also decrease the incidence of cerebral palsy and cognitive impairment of premature infants.The protection of caffeine citrate for central nervous system results form direct effects on brain or indirectly by improving the ventilation,the specific mechanism is still unclear. 7-day-old Sprague-Dawley rats were choosen for the experiment, animal model were established by the method of hypoxia-ischemia,and then the rats were given the teatment of CC by intraperitoneal injection,authenticated the neuropertective role of CC by observing the effect of CC on the expression of neuronal proliferation and apoptosis, myelin basic protein,the ability of long term learning and memory,and the changes on adenosine A1 receptors mRNA and A2 receptor mRNA after hypoxic-ischemic,and explore the possible mechanism of neuroprotective effect of CC in rats with hypoxic-ischemic.Materials and Methods72 7-day-old SD rats were randomly divided into three groups:sham group,HIBD group, CC group,each group had 24 rats. The rats in sham-operated group were only performed operation of separating the left common carotid artery,without of ligation or hypoxic.The HIBD animal model with reference to Rice,in the other two groups,the left common carotid artery of rats were ligated,and rats were returned to their dam for recovering 1 hours,then rats were placed in an airtight container with temperature at 37℃,the gas mixture flow to chamber was 80ml/L oxygen and 920ml/L nitrogen for 2 hours. The rats in CC groups were injected intraperitoneally CC with dosage of 20mg/kg,before administration of HI and at HI 0 min,24h,48h,72h, the rats in sham operation group and HIBD group were given the same volume saline by intraperitoneal injection at the same time.(1).On postnatal day 12,each group was randomly selected 16 rats for sacrifice,the other rats were left for testing Y maze on on postnatal day 28.Before postnatal day 12,8 Rats were randomly choosen for injecting with 5-bromodeoxyuridine (BrdU) at the dosage of 50mg/kg,once 12h,for consective 5 times,before last injection the rats were put to death. The expression of BrdU in the hippocampal dentate gyrus,Cleaved Caspase-3 in hippocampal CA1 area,and Subcortical white matter myelin basic protein of the injured brain were measured by immunohistochemistry,and neuronal apoptosis in hippocampal area CA1 area were detected by TUNEL method. The pathological changes of brain damage in each group was used by HE dyeing.(2).On postnatal day 12,8 rats were randomly choosen for sacrifice,the changes of AIR mRNA and A2a mRNA after CC in neonatal rats with HIBD were detected by Real Time PCR. (3).The remained 8 rats were used to conduct Y maze test for the ability of learning and memory on postnatal day 28.Results1. Pathological results of newborn rats brain tissue with HI in each groupHE staining of the sham operation group rats, hippocampal CA1 area in brain with hypoxia ischemia showed normal structure,clear layers. Brain tissue had different extents of pathological changes after hypoxia ischemia,nerve cell deranged,and uncleared structure. The hippocampal pyramidal cell layer,cell shape and arrangement in CC group were generally normal.2. The expression of BrdU in the hippocampal dentate gyrus,Cleaved Caspase-3 in hippocampal CA1 area,TUNEL positive cell in hippocampal CA1 area and Subcortical white matter myelin basic protein in neonatal rats brain of HI sideThe BrdU expression in the hippocampal dentate gyrus of hypoxia ischemia side of HIBD group(56.5±0.91) and CC group(59.1±0.54) were higer compared with the sham operation group(41.25±1.27) (P<0.05), but there were no statistically difference between the two groups(P>0.05).The Cleaved Caspase-3 expression in hippocampal CA1 area of hypoxia ischemia side of each group from high to low were HIBD group(52.38±1.60),CC group(25.88±0.64) and sham operation group(14.00±0.27) (P<0.05),there were statistical significance(P<0.05).The TUNEL positive cell expression in hippocampal CA1 area of hypoxia ischemia side of each group from high to low were HIBD group(37.38±0.94),CC group(14.75±0.56) and sham operation group(9.13±0.35) (P<0.05). The expression of subcortical white matter myelin basic protein in hypoxia ischemia side of HIBD group were lower than sham operation group(P<0.05),but the expression were higher when geven CC(P<0.05).3. The AIR mRNA and A2aR mRNA expression in neonatal rats brain of HI sideReal Time PCR showed that the AIR mRNA expression in brain with HI was significantly increased in HIBD group than sham operation group(1.28±0.34 vs 1.02±0.18)(P<0.05),but the changes of A2aR mRNA were not higher(1.13±0.36 vs 1.09±0.21)(P>0.05). The AIR mRNA expression was significantly decreased when rats given CC(0.94±0.17 vs 1.28±0.34)(P<0.05),but there were not clear changes inA2aR mRNA expression(0.96±0.14 vs 1.13±0.36)(P>0.05).4. The performance of long term learning and memory in neonatal ratsOn postnatal day 28,Y maze showed the learning number of avoiding electric shock from low to high order was sham operation group(31.25±1.41),(CC group(39.75±2.51) and HIBD group(58.50±3.14),significant differences existed in three groups(P<0.05).The ability of memory from high to low were CC group(58.50±3.14),sham group(83.75±3.24)and HIBD group(56.25±3.24),there are significant differences between HIBD group and the other two groups(P<0.05),but there showed no statistically significant differences between the first two groups(P>0.05).Conclusions1. Caffeine citrate had showed protective effect on HIBD,it could reduce neuronal apoptosis and inprove brain white matter damage.2. The protection of caffeine citrate in neonatal rats with HIBD might have relationships with down-regulate the expression of AIR.3. The treatment of caffeine citrate can improve the ability of long term learning and memory in neonatal rats with HIBD. |
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