Glucose transporters, hexokinase, and phosphofructokinase in brain of rats with perinatal asphyxia

Transport by glucose transporters from blood to the brain during hypoxic-ischemic conditions is well studied. However, the recent availability of a clinically related animal model of perinatal asphyxia and the fact that no concomitant determination of glucose transporters, parameters for glucose utilization, brain glucose, and cerebral blood flow (CBF) have been reported and the early phase of perinatal asphyxia has never been studied led us to perform the following study. Cesarean section was performed on full-term pregnant rats. The obtained pups within patent uterus horns were placed into a water bath at 37 degrees C from which they were subsequently removed after 5-20 min of graded asphyxia. Brain pH, brain tissue glucose, CBF, mRNA and activity of hexokinase and phosphofructokinase, and mRNA and protein of the glucose transporters GLUTI and GLUT3 were determined. Brain pH decreased and brain tissue glucose and CBF increased with the length of the asphyctic period; hexokinase and phosphofructokinase mRNA and activity were unchanged during the observation period. The mRNA and protein of both glucose transporters were comparable between normoxic and asphyctic groups. We show that glucose transport and utilization are unchanged in the early phase of perinatal asphyxia at a time point when CBF and brain glucose are already significantly increased and severe acidosis is present.     

Protein synthesis by gastric mucosal ribosomes during development in rats

Oxyntic gland mucosal ribosomes from 7- to 30-day-old rats were assayed for their ability to synthesize endogenous messenger RNA (mRNA) and poly(U)-directed protein in a cell-free system. Normal rat liver cell sap (105,000 g supernatant) was used as a source for transfer RNA (tRNA) and activating enzymes. Total mucosal DNA and ribosome content [as assessed by ribosomal RNA (rRNA)] were also determined. Although both DNA and rRNA content increased throughout the 4 postnatal weeks, the rRNA/DNA ratio rose sharply during the 2nd and 4th week after birth. The ability of mucosal polyribosomes to synthesize endogenous mRNA-directed protein in a cell-free system remained elevated up to the age of 18 days, then decreased markedly within the next 3 days, and thereafter, declined more slowly. In 21- and 30-day-old rats, the rate of endogenous mRNA-directed protein synthesis by mucosal ribosomes was 50-60% below that of the 7-day-old suckling rats. The protein/polyphenylalanine ratio, which represents a ratio of polysomes to monosomes, was found to be about 57% lower for ribosomes from 30-day-old rats as compared to 7-day-old suckling animals. The ability of run-off ribosomes (devoid of endogenous mRNA) to translate poly(U) was also found to be 33% lower for 30-day-old rats as compared to the 7-day-old suckling animals.     

Bid基因在新生大鼠缺氧缺血性脑损伤中的动态变化

目的　探讨Bid基因在新生大鼠缺氧缺血性脑损伤 (HIBD)中的动态变化。方法　通过建立新生大鼠HIBD动物模型 ,采用RT PCR技术检测缺氧缺血后不同时间点缺血侧脑组织中Bid基因表达的变化。结果　正常组中无Bid基因表达 ,缺氧缺血组Bid基因表达明显上调 ,且随着缺氧缺血后时间的延长 ,Bid基因表达呈动态变化缺氧缺血后 6h其表达开始增加 ,2 4h达高峰 ,48～ 72h有所回落。结论　脑缺氧缺血引起的神经细胞凋亡可能与Bid基因表达上调有关。     

Serum CPK–BB Isoenzyme in the Assessment of Brain Damage in Asphyctic Term Infants

ABSTRACT. Serum creatine phosphokinase-BB activities were measured at 4 and at 10 hours of life in 33 asphyctic full-term infants. The infants were followed for a mean period of 16 months. Enzyme activities at 4 hours of life were significantly higher in those neonates who died of severe hypoxic-ischemic encephalopathy or developed neurological sequelae than in those who did not present neurological abnormalities during the follow-up time. The specificity of the enzyme assay in order to predict the neurological outcome was inferior to that of cranial computed tomography or a combined clinical-electroencephalographic evaluation. It appears that the presence of elevated serum creatine phosphokinase-BB activity can be a sensitive indicator of conspicuous brain damage, but it is of limited value to predict the neurological outcome after neonatal asphyxia.     

Serum CPK-BB isoenzyme in the assessment of brain damage in asphyctic term infants

Serum creatine phosphokinase-BB activities were measured at 4 and at 10 hours of life in 33 asphyctic full-term infants. The infants were followed for a mean period of 16 months. Enzyme activities at 4 hours of life were significantly higher in those neonates who died of severe hypoxic-ischemic encephalopathy or developed neurological sequelae than in those who did not present neurological abnormalities during the follow-up time. The specificity of the enzyme assay in order to predict the neurological outcome was inferior to that of cranial computed tomography or a combined clinical-electroencephalographic evaluation. It appears that the presence of elevated serum creatine phosphokinase-BB activity can be a sensitive indicator of conspicuous brain damage, but it is of limited value to predict the neurological outcome after neonatal asphyxia.     

神经节苷脂对脑缺氧缺血后新生大鼠海马CA1区 N-甲基-D-天门冬氨酸受体表达的影响

目的 研究新生大鼠缺氧缺血性脑损伤(HIBD)后海马区N-甲基-D-天门冬氨酸受体(NMDAR)及NMDARmRNA表达的变化和神经节苷脂(GM1)对其的影响。方法 建立HIBD模型。用免疫组化及原位杂交方法检测缺氧缺血(HI)和GM1干预后不同时间点NMDA受体I型亚单位(NR1)阳性细胞及NR1mRNA阳性细胞的表达。结果 HI后新生大鼠海马CA1区NR1及NR1mRNA表达增强,GM1可使其受体表达下调(P<0.05)。结论 GM1可使新生大鼠海马CA1区NR1及NR1mRNA表达下调,对新生大鼠缺氧缺血性脑损伤具有保护作用。     

新生大鼠缺氧缺血性脑病模型脑组织新生血管形成及调控因素

目的探讨新生大鼠缺氧缺血性脑病(HIE)模型脑组织新生血管形成及调控因素.方法 68只新生大鼠建立HIE模型组(44只)或行假手术组(24只).两组缺氧后1、3、7和14天各处死5只大鼠,用免疫组化方法检测脑组织内皮细胞、增生细胞核抗原和血管内皮生长因子(VEGF).模型组的其余24只新生大鼠建立HIE模型,于缺氧后3、12h,1、3、7和14d各处死4只大鼠,取缺氧缺血侧脑组织液氮速冻,用于提取RNA.假手术组的其余4只正常新生大鼠作假手术处理后即刻处死提RNA做正常对照.分析缺氧缺血侧脑组织毛细血管密度(BCDI)、增生毛细血管密度和VEGF表达.用RT-PCR检测VEGF和缺氧诱导因子-1α (HIF-1α) mRNA表达.结果模型组缺氧缺血侧脑组织坏死灶周围BCDI在第3天开始上升,并持续增加;BCDI显著高于假手术组(第14天13.29±3.90条/HPF vs 6.08±1.50条/HPF,P＜0.01).正常新生大鼠脑组织偶见增生毛细血管,而模型组缺氧缺血侧脑组织增生毛细血管密度在第3、7天(0.54±0.15条/HPF和0.90±0.25条/HPF)显著高于假手术组(0.12±0.05条/HPF和0.13±0.07条/HPF,P＜0.01).VEGF主要由神经元、毛细血管内皮细胞和软膜细胞等细胞表达.缺氧缺血后脑组织VEGF mRNA表达上调,12h时VEGF mRNA表达显著高于正常(1.56±0.27 vs 0.95±0.21, P＜0.05);HIF-1α mRNA表达上调先于VEGF,3h时HIF-1α mRNA表达即显著高于正常(1.07±0.21 vs 0.64±0.28, P＜0.05).结论新生大鼠HIE模型脑组织有新生血管形成; HIF-1介导的VEGF表达上调在此过程中可能起重要作用.     

地塞米松对脑缺氧缺血新生大鼠细胞凋亡抑制蛋白1 mRNA及Caspase-3活性的影响

目的：探讨新生大鼠缺氧缺血性脑损伤（HIBD）后细胞凋亡抑制蛋白1（cIAP1）基因表达、Caspase-3活性的变化及地塞米松（DEX）对其影响，阐明DEX预处理对HIBD神经保护的可能机制。方法：7日龄SD大鼠24只，随机分成DEX组、9g／L盐水组（NS组）、HIBD组、健康对照组。DEX、NS、HIBD组大鼠采用Rice方法制备HIBD模型。DEX和NS组在缺氧缺血前12h腹腔内分别注射DEX、等量9g／L盐水。取HIBD动物模型实验侧大脑半球，采用逆转录-聚合酶链反应（RT-PCR）检测cIAP1基因表达，比色法测定Caspase-3活性。结果：与健康对照组比较，HIBD组大鼠cIAP1基因表达明显下降（P〈O．01），Caspase-3活性明显上升（P〈0．01）。与HIBD组比较，DEX组cIAP1基因表达明显上升，而Caspase-3活性明显下降。结论：脑缺氧缺血可导致cIAP1基因表达下调，Caspaes-3活性升高。DEX能通过上调cIAP基因表达，抑制Caspase-3活性，抑制细胞凋亡，起到神经保护作用。     

碱性成纤维细胞生长因子对缺氧缺血性脑损伤新生鼠骨形态发生蛋白4及其mRNA表达的影响

目的 探讨碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对新生鼠HIBD骨形态发生蛋白4蛋白及其mRNA表达的影响.方法 新生7日SD乳鼠120只,随机分①bFGF组、②HIBD组、③正常对照组,每组40只.HIBD模型建立后①组予以bFGF干预,腹腔注射,连用5 d,根据处死时相点各组又分为7、14、21、28 d等4个小组.采用免疫组化技术检测各组骨形态发生蛋白4(BMP4)蛋白在海马的表达;采用原位杂交技术检测各组BMP4 mRNA在海马的表达;采用TUNEL实验检测各组神经细胞的凋亡.采用随机组设计资料的方差分析进行组间及组内比较.结果 在7 d和14 d时相点,bFGF组BMP4蛋白在海马的表达较H1BD组多;2组在14 d时相点BMP4蛋白在海马的表达较7 d时相点弱.21 d小组、28 d小组等BMP4蛋白在海马的表达3组之间数量无明显变化.在7 d时相点,HIBD组、bFGF组CA1区BMP4 mRNA表达明显,较正常对照组明显增加;在14 d时相点,HIBD组BMP4 mRNA在病侧海马广泛表达,bFGF组仅病侧海马CA1区BMP4 mRNA表达明显,但2组较正常对照组明显增加;21 d时相点BMP4 mRNA在HIBD组、bFGF组海马的表达均显著;28 d时相点BMP4 mRNA在HIBD组病侧海马的表达开始减少,而bFGF组BMP4mRNA在病侧海马的表达无变化.bFGF组凋亡神经细胞在7 d时相点较HIBD组少[(20.10±0.35)vs(29.12±0.31);F=9.010,P<0.01];在14、21、28 d 3个时相点,HIBD组和bFGF组凋亡神经细胞均较7 d时相点增多,bFGF组凋亡神经细胞仍较HIBD组少[(28.09±0.26) vs (37.46±0.23);(18.75±0.71) vs (35.36±0.77);(12.26±0.57) vs (25.70±0.21);F=9.202,7.932,14.985,P<0.01].结论 bFGF对HIBD具有神经修复作用,其作用机制足促进BMP4蛋白及其mRNA在新生鼠HIBD海马的表达,并抑制神经细胞的凋亡.     

缺氧缺血新生大鼠神经元自噬基因表达节律及调控机制

目的通过新生大鼠缺氧缺血脑损伤后神经元自噬基因和节律基因的表达,探讨缺氧缺血引起神经损伤的新机制。方法将12只Sprague-Dawley大鼠随机分为缺氧缺血组和假手术组,每组6只。采用结扎并切断大鼠右侧颈总动脉并给予低氧处理的方法建立体内缺氧缺血脑损伤模型。Western blot检测两组大鼠大脑皮层和海马组织节律蛋白Clock表达情况。体外培养大鼠神经元细胞,随机分为氧糖剥夺(OGD)组和对照组,OGD组加入无糖无血清DMEM培养基模拟细胞缺血状态,并给予低氧处理建立体外缺氧缺血脑损伤模型。采用Western blot检测两组不同时间点自噬相关蛋白Beclin1和LC3,以及节律基因Clock蛋白表达情况。应用si RNA技术抑制神经元Clock蛋白表达后,检测Beclin1和LC3的表达变化。结果体外培养神经元Beclin1和LC3Ⅱ的表达呈现节律性波动;OGD处理后,体外培养神经元Beclin1和LC3Ⅱ的表达随着时间的延长逐渐升高,不再呈现节律性波动;与假手术组相比,缺氧缺血引起大鼠皮层和海马组织Clock表达降低(P0.05);体外培养神经元经OGD处理后,Clock表达也较对照组显著降低(P0.05);与阴性对照组相比,抑制神经元节律基因Clock表达后,自噬相关蛋白Beclin1和LC3Ⅱ的表达均显著下降(P0.05)。结论缺氧缺血引起神经元Beclin1和LC3Ⅱ表达节律紊乱,其机制可能与Clock参与调控Beclin1和LC3Ⅱ的表达有关。     

Reduction of hypoxic-ischemic brain swelling in the neonatal rat with PAF antagonist WEB 2170: lack of long-term protection

Platelet activating factor (PAF) is an inflammatory lipid mediator released by ischemic brain. Our objectives were to use an inhibitor of PAF that does not readily cross the blood-brain barrier, WEB 2170, to study the role of intravascular PAF on brain swelling and subsequent brain atrophy in a neonatal rat model of hypoxic-ischemic brain injury. We injured the right cerebral hemisphere of 7-d-old rats by ligating the right common carotid artery and exposing the rats to 8% oxygen for 2.25 h. Forty-two rats received saline or the PAF antagonist WEB 2170, 1 h before hypoxia. We found that WEB 2170 pretreatment reduced swelling by 64% (p = 0.003). In contrast, treatment immediately after hypoxic-ischemic injury did not reduce swelling. In two additional experiments involving 103 rats, we found that pretreatment or repeated doses of PAF antagonist before and after hypoxic-ischemic injury did not reduce atrophy. We also found that the brain-penetrating PAF antagonist, BN 52021, did not prevent atrophy in our Wistar rat model. In conclusion, we were unable to reduce long-term brain injury with either PAF antagonist. WEB 2170 pretreatment reduced brain swelling by 64% without reducing atrophy. This suggests that although brain swelling may accompany cerebral infarction, it does not contribute to the pathogenesis of infarction and subsequent atrophy in the neonatal rat. The ability to reduce early postischemic brain swelling without reducing atrophy may be particularly unique to the immature animal with a compliant skull.     

神经生长因子对新生大鼠缺氧缺血性脑损伤后神经元特异性烯醇化酶的影响

目的 研究神经生长因子（NGF）对新生大鼠缺氧缺血性脑损伤（HIBD）的作用。方法 应用新生7日龄SD大鼠制备HIBD模型,于缺氧缺血（HI）后即刻腹腔注射NGF 100U／只或等量生理盐水,用放射免疫法测定缺氧缺血后24、48h血清神经元特异性烯醇化酶（NSE）水平,用免疫组化法检测脑组织NSE蛋白表达变化。结果 NGF可降低血清NSE,增加左侧（缺血侧）脑组织中NSE蛋白水平表达。结论 腹腔注射NGF对HIBD具有保护作用。     

Effects of Pulmonary Regurgitation on Distensibility and Flow of the Branch Pulmonary Arteries in Tetralogy of Fallot

Significant pulmonary regurgitation (PR) after repair of tetralogy of Fallot (TOF) may affect flow in the pulmonary artery (PA) side branches. We sought to assess flow changes and distensibility of the PA side branches in vivo and test correlation with the degree of PR and right-ventricular (RV) dilatation. Thirty patients after TOF repair and 16 controls underwent cardiovascular magnetic resonance for quantification of RV volumes and measurement of flow in the PA side branches. RV volumes and function, blood flow volumes, and cross-sectional area of the main, left (LPA), and right (RPA) PA were measured and regurgitant volumes and distensibility calculated. Results were compared between the LPA and the RPA and between patients and controls. Median regurgitation fraction of PR was 41 % (range 22–60 %). Regurgitant fraction was greater in the LPA (40 %) than in the RPA (29 %), resulting in lower net flow into the LPA (p < 0.001). LPA area was significantly greater than that of the RPA (303.9 vs. 232.7 mm²/m²) (p < 0.0001). The LPA showed lower distensibility than the RPA (39 vs. 44 %). PA side branch distensibility correlated with MPA regurgitant volume (p = 0.001), MPA regurgitant fraction (p = 0.001), and RV end-diastolic volume (p = 0.03). PA side branches have greater distensibility in patients with PR than in normal subjects. Significant PR leads to changes in flow profile and distensibility of the PA side branches. The LPA shows greater regurgitant volume and greater area but lower distensibility than the RPA.     

缺氧缺血后新生鼠脑组织caspase-3mRNA表达变化及其意义

目的　观察新生大鼠缺氧缺血后脑组织caspase 3mRNA表达动态变化 ,进一步探讨缺氧缺血诱导新生鼠神经元凋亡的机制及意义。方法　制备新生鼠HIBD模型 ,随机分为假手术对照组、HIBD 6h、2 4h、3d、5d组 ,每组 5只动物。应用RT PCR方法测定了大鼠脑组织caspase 3mRNA的表达。结果　 7日龄Wistar大鼠假手术对照组即有一定水平caspase 3mRNA表达 ,HIBD 6h组表达增加(与对照组比较有显著性差异 ,P <0 0 5 ) ,HIBD 2 4h其表达呈高峰 ,此后逐渐下降 ,HIBD 5d仍维持较高水平 (与HIBD 6h相比有显著性差异 ,P <0 0 1)。结论　缺氧缺血可诱导新生鼠脑组织caspase 3mRNA表达 ,其过度表达在HIBD后神经元凋亡的调控中起一定作用 ,针对caspase 3的治疗对策为围生期缺氧缺血脑损伤打开了新视窗     

Orotic acid: its influence on nuclear RNA and microsomal protein synthesis in brain cells of undernourished rat pups

Malnourished rat pups were supplemented with orotic acid. RNA-synthesizing activity of isolated brain cell nuclei and microsomal protein synthesis was measured in vitro. A marked increase was found in the activity of nuclear RNA synthesis but not in microsomal protein synthesis after orotic acid treatment for 7 days.     

碘标记神经生长因子在缺氧新生鼠脑内的吸收

目的观察碘标记神经生长因子(125I-NGF)在缺氧新生鼠脑内的吸收情况。方法选取32只7日龄健康新生鼠随机分成实验组(125I-NGF组和125I组)及正常组(125I-NGF组和125I组),实验组制成缺氧缺血性脑损伤(HIBD)模型。分别予125I-NGF和单纯125I腹腔注射后30 min处死取脑,按如下解剖分出基底前脑、额皮质、海马、丘脑下区、小脑、嗅球、垂体,对标本进行放射性测量。结果两125I NGF组于基底前脑、小脑、额皮质、海马、嗅球均有显著吸收,且NGF在缺氧鼠额皮质、海马、小脑吸收显著高于NGF在正常新生鼠以上部位。结论外源性NGF可透过新生鼠血-脑脊液屏障,早期应用治疗HIE是可行的。     

肌苷对新生大鼠缺氧缺血性脑损伤神经细胞凋亡和细胞色素C基因表达的影响(英文)

目的：目前认为神经细胞凋亡在新生儿缺氧缺血性脑病(hypoxicischemicencephalopathy,HIE)的病理过程中起重要作用,细胞色素C(CytC)是重要的促凋亡蛋白因子之一。近年研究发现,肌苷对成年大鼠脑缺血损伤有保护作用,肌苷可降低CytCmRNA的表达从而抑制神经细胞凋亡。本实验通过观察肌苷对新生大鼠缺氧缺血性脑损伤(HIBD)后神经细胞凋亡和细胞色素C基因表达的影响,以初步探讨肌苷对HIBD新生大鼠脑保护作用和可能的机制。方法：健康7日龄SD大鼠140只被随机分为3组:正常对照组(n=40),肌苷治疗组(n=50)和HIBD组(n=50),其中肌苷治疗组和HIBD组分别再随机分为缺氧缺血(HI)后6h,12h,1d,3d,7d5个亚组(各亚组n=10)。通过分离、结扎左颈总动脉和8%低氧暴露制备HIBD动物模型。正常对照组不进行缺氧缺血处理,按肌苷治疗组和HIBD组各相同时间点随机分为5个亚组(各亚组n=8)。肌苷治疗组于模型制备后即刻开始腹腔注射肌苷注射液100mg/kg,每天2次,连续7d。以TUNEL法检测神经元凋亡情况,原位杂交技术测定CytCmRNA表达情况。结果：正常对照组皮质区和海马区可见少许凋亡细胞和CytC阳性细胞,HI后6hHIBD组皮质区和CA1区凋亡细胞和CytC阳性细胞即见增多,于HI后1d达高峰,之后逐渐下降,HI后7d凋亡细胞和CytC阳性细胞数仍明显高于正常对照组,各时间点与正常对照组相比较,差异有显著性意义(P0.05)。经肌苷治疗后凋亡细胞数和神经细胞CytCmRNA表达均减少,各时间点与HIBD组相应时间点比较,差异有显著性意义(P0.05)。直线相关分析显示HIBD后,凋亡细胞数与CytCmRNA表达呈显著正相关(r=0.88,P0.01)。结论：HI损伤后给予肌苷干预能减少HI导致的神经细胞凋亡,下调CytCmRNA表达。肌苷治疗后,新生HIBD大鼠的凋亡细胞数减少与CytCmRNA表达下调呈显著正相关,提示肌苷可能通过抑制CytCmRNA表达从而起到减少细胞凋亡、保护神经元的作用。